Method of inducing a CTL response

ABSTRACT

Disclosed herein are methods for inducing an immunological CTL response to an antigen by sustained, regular delivery of the antigen to a mammal so that the antigen reaches the lymphatic system. Antigen is delivered at a level sufficient to induce an immunologic CTL response in a mammal and the level of the antigen in the mammal&#39;s lymphatic system is maintained over time sufficient to maintain the immunologic CTL response. Also disclosed is an article of manufacture for delivering an antigen that induces a CTL response in an animal.

CROSS REFERENCE

[0001] This application is a continuation-in-part of U.S. PatentApplication No. 09/380,534, filed Sep. 1, 1999, which was based on PCTApplication No. PCT/US98/14289, filed Jul. 10, 1998, which claimedpriority from U.S. Patent Application No. 08/988,320, filed Dec. 10,1997, and from Canadian Patent Application No. 2,209,815, filed Jul. 10,1997, all of which are hereby incorporated by reference in theirentirety.

FIELD OF THE INVENTION

[0002] The invention relates to a method of inducing a CTL response toan antigen by sustained, regular delivery of the antigen to an animal sothat the antigen reaches the lymphatic system.

BACKGROUND OF THE INVENTION

[0003] Cytotoxic T lymphocytes (CTL) are white blood cells found in theblood, spleen and lymph. CTL have the ability to attack and kill othercells of the body in a highly specific manner. When CTL are stimulatedby specific antigen, they migrate through the tissues of the body on a“search and destroy” mission for cells bearing the specific antigen.Whether of viral origin or tumor associated, CTL detect antigen that isbound to major histocompatability complexes (MHC) on the surface ofpotential target cells. Once CTL have identified the antigen on the cellsurface, their function is to deliver a lethal hit to the cell.

[0004] Although there are hundreds of millions of CTL that reside in thespleen, each individual CTL exclusively responds to a unique andspecific antigen. These individual CTL, dubbed CTL precursors (CTLp),undergo cell division or proliferate upon activation by specific antigento produce daughter cells with precisely the same antigen specificity asthe parent cell. This proliferation increases the total number, and thusthe frequency, of that specific CTLp in the body. A proportion of thesenewly generated CTL briefly recirculate through the body (termedeffector CTL), and have the ability to identify and destroy cellsbearing the specific antigen which they recognize. A significant body ofexperimental evidence suggests that CTL specific for tumor antigens caninhibit tumor growth. Unfortunately, most tumors have only a very weakcapacity to stimulate CTL responses and there has been no means ofinducing a CTL response then sustaining it over a period of timesufficient to continuously inhibit tumor growth. While many attempts todirectly increase the capacity of tumor cells to stimulatetumor-clearing CTL responses in patients have been made, such attemptshave met with limited success. Technical advances over the past tenyears have, however, enabled the identification of natural peptideantigens that are present on tumor cells and which are recognized byCTL. These antigen targets include proteins expressed in significantoverabundance, abnormally expressed embryonic proteins, protein productsfrom mutated oncogenes or suppressor genes, or proteins derived fromcancer-causing viruses present in tumor cells. The challenge has been tofind a way in which to administer an antigen so that it induces anantitumor CTL response and maintains it over time. While many attemptshave now been made to use these antigens clinically in a vaccine, theresults have been less than satisfactory.

[0005] An explanation of why CTL therapies have been largely ineffectiveat eradicating or controlling tumors in a clinical setting include thefollowing:

[0006] (a) Vaccine designs have been inadequate at initiating strong CTLresponses;

[0007] (b) Tumor cells can down regulate MHC molecules, resulting in theloss of antigen presentation from the surface of cells, thereby escapingdetection by CTL;

[0008] (c) After induction, effector CTL recirculation through the bodyis highly transient;

[0009] (d) After recirculation, CTL return to the spleen where theyreside in a nonactive or resting state, and an increase in the numbersof CTLp residing in the spleen does not reflect active CTL immunity;

[0010] (e) In the case of tumors, regrowth of residual tumor cellsfollowing immunization goes undetected by CTLp residing in spleen in a“resting” state;

[0011] (f) Because CTL-stimulating antigen presenting cells (APC aretargeted for destruction by the same CTL that they have activated, theCTL response is self-limiting, which precludes, under normalcircumstances, the continuous stimulation for a long-lived CTL response.

[0012] A growing repertoire of tumor associated antigens are beingdiscovered that are recognized by CTL. A variety of techniques have beensuggested to render these antigens effective in CTL vaccines. Theseinclude immunization using synthetic peptide antigens mixed with animmunostimulatory adjuvant, such as the bacterial toxin BCG;inununization with multiple antigenic peptide systems (MAPS),immunization with “professional” antigen presenting cells, which areisolated from the patient, pulsed with peptide antigen and inoculatedback into the patient as a vaccine; immunization with peptides designedto stimulate both CTL and T helper cell populations; immunization withviruses or bacteria engineered to express tumor antigens; andimmunization with polynucleotide expression vectors (so called DNAvaccines). Unfortunately, none of these approaches has been anunqualified success, As discussed above, the lack of vigoroustherapeutic effects with these vaccine platforms reflects at least tosome degree problems associated with inducing a strong initial CTLresponse and with maintaining ongoing “active” CTL immunity.

[0013] Studies by Glenny during the first quarter of the centuryrevealed that aluminum compounds could enhance the strength ofdiphtheria vaccines. This was ostensibly the first of a long history ofobservations supporting a “depot” theory of immunization, whichpostulates that antigen slowly leaking into the tissues over an extendedtime correlates with the antigenic potency of a vaccine. Today, thisantigen depot paradigm forms the intellectual backdrop to most adjuvantdevelopment programs. In one form or another, depot type adjuvants areintended to prolong the course of antigen delivery, by forming a lesionat the site of injection, or simply by the slow degradability of theadjuvant itself, which mixed with the specific antigen forms a depot atthe site of injection. A second function generally attributed toadjuvants are their immunostimulatory effects, which appears to triggerthe immune system to respond to the vaccine. However, adjuvants are adouble-edged sword. They have inherent toxicities. But it is a featureof these toxicities that achieves a desired immunostimulatory and/ordepot effect. Side effects such as tissue damage and granulomatousreaction at the site of injection, fever, and in some cases systemicreactions, such as Reiter's syndrome-like symptoms, uveitis andarthritis, are some of the risks associated with the use of adjuvants.Currently, the only adjuvant approved by the FDA is alum. It isrelatively safe but does have side effects such as erythema,subcutaneous nodules, contact hypersensitivity, and granulomatousinflammation. More importantly, alum only acts to potentiate a limitednumber of antigens, and it very predominantly stimulates humoralantibody responses rather than CTL immunity. Thus so far adjuvants haveproved to be very ineffective components for vaccines aimed at inducingclinically relevant CTL responses.

[0014] Recent attempts to induce CTL responses using dendritic cells orother antigen presenting cells, despite being cumbersome, have shownsome promise. New recombinant virus or bacterial systems carrying genesfor specific antigen are effective at inducing primary CTL responses.The most effective viruses, for example, that induce strong CTLresponses are those which replicate aggressively in the host. Yetbecause of the risk for serious or lethal complications as a result ofinfection, recombinant virus used in a cancer vaccine must be onlyweakly replicative, or be completely replication deficient. Thistrade-off between virulence and efficacy is at present an intractableproblem.

[0015] DNA (or polynucleotide) vaccines are also being developed for thepurpose of inducing CTL immunity. Once again, the system has intrinsiclimitations that preclude its efficacy in inducing long-lasting CTLimmunity. The DNA vaccines consist of a plasmid or similar geneticconstruct for expressing the antigen of interest. Uptake of the plasmidsystem by cells of the body results in expression of the antigen andinduction of CTL. However, once cells expressing the construct havesucceeded in inducing CTL, they are themselves targets for eradicationby the CTL. The CTL inducing effect is thus again transient. Moreover,the polynucleotide vaccines have thus far suffered from poor efficiencyin terms of CTL induction.

[0016] With difficulties in achieving strong primary and/or persistingCTL responses, there are a number of clinical trial groups now usingrepeated injections of cancer vaccines. The use of antigenically complexmaterials in the vaccine formulation, such as recombinant virus, or thecosts associated with repetitive treatment using cultured APC will,however, make such an approach difficult. On the one hand, repetitiveimmunization with antigenically complex materials drives the immunesystem to elaborate a humoral antibody, as opposed to a CTL response,while on the other hand, use of a minimal CTL antigen (such as a nonamerpeptide) which does not efficiently drive an antibody response, has alsofailed to induce a CTL response. Attempts to develop adjuvants thatenhance the immunostimulatory aspects of minimal CTL antigens haveresulted in the production of materials (i.e. adjuvants) that alsoinduce a competing humoral immune response, or, which simply offerlittle CTL stimulatory effect.

[0017] It has also been suggested that certain controlled releasetechnology using microspheres or liposomes with subunit antigens andpeptides might be effective to enhance immunogenecity. The combinationof sustained release and depot effect is suggested to reduce the amountof antigen needed and eliminate booster shots. However, the preparationof such compositions is difficult and unpredictable, and vaccineformulations based on this technology have not been translated intoeffective clinical treatments.

[0018] As can be seen from the foregoing, there has been little successat developing a CTL vaccine that is both capable of inducing a strongCTL response then sustaining that response over time. The development ofa vaccine with these capabilities is essential before effectiveanti-tumor therapy based on CTL immunity can be contemplated.

OBJECTS OF THE INVENTION

[0019] An object of this invention is to provide a method for inducingor sustaining a specific CTL immununological response in a mammal overtime.

[0020] Another object of this invention is to provide a method fortreating a mammal having a malignant tumor or infectious disease byinducing and sustaining an immunological attack on the malignant tumoror infectious disease in the mammal.

[0021] It is a further object of this invention to provide an article ofmanufacture useful for inducing and sustaining a specific immunologicalCTL response in a mammal over time.

[0022] It is a further object of this invention to provide an article ofmanufacture useful for treating a mammal having a malignant tumor orinfectious disease, which article is designed to induce and maintain animmunological attack on the malignant tumor or infectious disease in themammal.

[0023] It is a further object of this invention to provide a portabledevice for sustained delivery of an antigen to a mammal having amalignant tumor or infectious disease, where the antigen stimulates themammal's immune system to attack the tumor or infectious disease and thedevice is located outside the mammal.

[0024] It is still a further object of this invention to provide animplantable device for sustained delivery of an antigen to a mammalhaving a malignant tumor or infectious disease, where the antigenstimulates the mammal's immune system to attack the tumor or infectiousdisease.

[0025] It is a further object of this invention to provide antigencompositions and containers therefor that are useful in the methods,devices, and/or articles of manufacture of this invention.

[0026] Other objects of this invention may be apparent to those of skillin the art by reading the following specification and claims.

SUMMARY OF THE INVENTION

[0027] In one aspect of the invention, a method is provided for inducingan immunological CTL response to an antigen by sustained, regulardelivery of the antigen to a mammal so that the antigen reaches thelymphatic system. In particular, the antigen is delivered to the mammalat a level sufficient to induce an immunologic CTL response in themammal and the level of the antigen in the mammal's lymphatic system ismaintained over time sufficient to maintain the immunologic CTLresponse. Preferably, the antigen is delivered directly to the mammal'slymphatic system, such as to the spleen, a lymph node or lymph vessel.

[0028] Also provided is a method of treating an animal having a disease,or being predisposed to a disease, to which the animal's immune systemmounts a cell-mediated response to a disease-related antigen to attackthe disease. In this aspect of the invention, a disease-matched antigenis delivered to the animal at a level sufficient to induce an increasedCTL-response in the animal which is then maintained in the animal bysustained, regular delivery of the disease-matched antigen to the animalfor a time sufficient to treat the disease. The sustained, regulardelivery of the antigen is done in a manner that maintains the level ofantigen in the animal's lymphatic system. Preferably, the sustained,regular delivery is achieved by pumping a physiologically-acceptable,composition of the antigen from a device held external of or implantedin the animal's body so that the antigen reaches the animal's lymphsystem. Optionally, a cytokine that is capable of enhancing the CTLresponse is delivered and/or maintained along with the antigen. Diseasesaddressed in this manner include cancer and pathogenic diseases.

[0029] In a further aspect of the invention, an article of manufactureis provided for delivering an antigen that induces a CTL response in ananimal. In particular, the article comprises a reservoir of aphysiologically-acceptable, antigen-containing composition that iscapable of inducing a CTL response in an animal; a pump connected to thereservoir to deliver the composition at a defined rate; a transmissionline to discharge the composition from the reservoir; and, optionally, adelivery line connected to the transmission line, which delivery line isof a size suitable for positioning in the animal and for delivery of thecomposition in a manner that reaches the lymphatic system of the animal.

[0030] In a further aspect of the invention, a process is provided forpreparing a system useful for inducing a sustained CTL response in ananimal needing such a response, which comprises placing aphysiologically-acceptable, antigen-containing composition in areservoir having a pump for delivering the composition at a defined ratethrough a transmission line to the animal.

[0031] Another aspect of the invention is a method of inducing and/orsustaining an immunological CTL response in a mammal by delivering anantigen in the form of a polypeptide directly to the lymphatic system ofthe mammal. The antigen can be delivered at a level sufficient to inducean immunologic CTL response in the mammal and the level of the antigenin the mammal's lymphatic system is preferably maintained over timesufficient to maintain the immunologic CTL response.

[0032] The antigen can be an 8-10 amino acid peptide. Further, thepeptide sequence can be derived from a tumor-associated antigen.Examples of tumor-associated antigens include MelanA (MART-I), gp100(Pmel 17), tyrosinase, TRP-1, TRP-2, MAGE-1, MAGE-3, BAGE, GAGE-1,GAGE-2, p15(58), CEA, RAGE, NY-ESO(LAGE), SCP-1, Hom/Mel-40, PRAME, p53,H-Ras, HER-2/neu, BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, EpsteinBarr virus antigens, EBNA, human papillomavirus (HPV) antigens E6 andE7, TSP-180, MAGE-4, MAGE-5, MAGE-6, p185erbB2, p180erbB-3, c-met,nm-23H1, PSA, TAG-72-4, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras,β-Catenin, CDK4, Mum-1, p16, TAGE, PSMA, PSCA, CT7, telomerase, 43-9F,5T4, 791 Tgp72, alpha-fetoprotein , β-HCG, BCA225, BTAA, CA 125, CA 15-3(CA 27.29\BCAA), CA 195, CA 242, CA-50, CAM43, CD68\KP1, CO-029, FGF-5,G250, Ga733 (EpCAM), HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB/70K,NY-CO-1, RCAS1, SDCCAG16, TA-90 (Mac-2 binding protein\cyclophilinC-associated protein), TAAL6, TAG72, TLP, TPS, and the like.

[0033] The peptide sequence also can be derived from a microbialantigen. Further, the antigen can be provided as a component of amicroorganism or mammalian cell. Examples of microorganisms include aprotozoan, a bacterium, a virus, and the like; the mammalian cell can bean antigen presenting cell, such as, for example, a dendritic cell.

[0034] The antigen can be a native component of the microorganism ormammalian cell. The microorganism or mammalian cell can include, forexample, an exogenous antigen. Also, the microorganism or mammalian cellcan include a recombinant nucleic acid encoding or promoting expressionof the antigen. The microorganism or mammalian cell can express atumor-associated antigen, or a microbial antigen native to a secondmicrobial species. The antigen can be provided as an 8-10 amino acidpeptide.

[0035] The present invention in another aspect includes a method ofinducing and/or sustaining an immunological CTL response in a mammal bydelivering an antigen, in the form of a vector that can include anucleic acid encoding the antigen, directly to the lymphatic system ofthe mammal. The antigen can be delivered at a level sufficient to inducean immunologic CTL response in the mammal and the level of the antigenin the mammal's lymphatic system is preferably maintained over timesufficient to maintain the immunologic CTL response.

[0036] The vector can be a plasmid and the like. The vector further caninclude a bacterium and the like. The bacterium, for example, caninclude Listeria, Shigella, Salmonella, Escherichia, and the like. Thevector, for example, can be a virus, such as, for example, pox viruses,adenoviruses, adeno-associated viruses, retroviruses, herpesviruses, andthe like.

[0037] The nucleic acid can encode, for example, a tumor-associatedantigen. Examples of tumor-associated antigens include MelanA (MART-I),gp100 (Pmel 17), tyrosinase, TRP-1, TRP-2, MAGE-1, MAGE-3, BAGE, GAGE-1,GAGE-2, p15(58), CEA, RAGE, NY-ESO (LAGE), SCP-1, Hom/Mel-40, PRAME,p53, H-Ras, HER-2/neu, BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR,Epstein Barr virus antigens, EBNA, human papillomavirus (HPV) antigensE6 and E7, TSP-180, MAGE-4, MAGE-5, MAGE-6, p185erbB2, p180erbB-3,c-met, mn-23H1, PSA, TAG-72-4, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras,β-Catenin, CDK4, Mum-1, p16, TAGE, PSMA, PSCA, CT7, telomerase, 43-9F,5T4, 791 Tgp72, alpha-fetoprotein , β-HCG, BCA225, BTAA, CA 125, CA 15-3(CA 27.29\BCAA), CA 195, CA 242, CA-50, CAM43, CD68\KP1, CO-029, FGF-5,G250, Ga733 (EpCAM), HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB/70K,NY-CO-1, RCAS1, SDCCAG16, TA-90 (Mac-2 binding protein\cyclophilinC-associated protein), TAAL6, TAG72, TLP, TPS, and the like.

[0038] The nucleic acid can encode, for example, a microbial antigen.Examples of microbial antigens include a viral antigen, a bacterialantigen, a protozoal antigen, and the like. The nucleic acid can encode,for example, a protein or other polypeptide. The nucleic acid also canencode an 8-10 amino acid peptide.

[0039] The nucleic acid can be plasmid DNA in a formulation comprisingabout 1-10% ethyl alcohol, 0-1% benzyl alcohol, 0.25-0.5 mM EDTA and acitrate-phosphate buffer of pH 7.4-7.8, comprising about 3-50 mM citrateand about 90 -200 mM phosphate. For example, the formulation can include1% ethyl alcohol, 1% benzyl alcohol, 0.5 mM EDTA and a citrate-phosphatebuffer of pH 7.4 to 7.8 comprising 50 mM citrate and 100 mM phosphate.

[0040] The invention in another aspect provides a method of inducingand/or sustaining an immunological CTL response in a mammal bydelivering a microorganism or mammalian cell directly to the lymphaticsystem of the mammal. The microorganism or mammalian cell are preferablydelivered at a level sufficient to induce an immunologic CTL response inthe mammal and the level of the microorganism or mammalian cell in themammal's lymphatic system is preferably maintained over time sufficientto maintain the immunologic CTL response.

[0041] A further aspect of the invention is a method of inducing and/orsustaining an immunological CTL response in a mammal by delivering anucleic acid, capable of conferring antigen expression, directly to thelymphatic system of the mammal. The nucleic acid can be delivered at alevel sufficient to induce an immunologic CTL response in the mammal andthe level of the nucleic acid in the mammal's lymphatic system ispreferably maintained over time sufficient to maintain the immunologicCTL response.

[0042] A further aspect of the invention is a method of inducing and/orsustaining an immunological CTL response in a mammal by delivering anon-peptide antigen directly to the lymphatic system of the mammal. Theantigen is preferably delivered at a level sufficient to induce animmunologic CTL response in the mammal and the level of the antigen inthe mammal's lymphatic system is preferably maintained over timesufficient to maintain the immunologic CTL response.

[0043] The invention also provides an article of manufacture fordelivering an antigen that induces a CTL response in an animal. Inparticular, the article can be an external device. The article caninclude a reservoir of a physiologically-acceptable, antigen-containingcomposition that can be capable of inducing a CTL response in an animal,a pump connected to the reservoir to deliver the composition at adefined rate, a transmission line to discharge the composition from thereservoir; and, a delivery line connected to the transmission line. Thedelivery line can include a catheter of at least 20 mm for positioningin the animal and for delivery of the composition to the lymphaticsystem of the animal.

BRIEF DESCRIPTION OF THE DRAWINGS

[0044] The invention will now be described in relation to the drawingsin which:

[0045]FIG. 1 is a graph showing the lysis of target cells by CTL versusthe effector/target ratio when antigen is delivered as a single dose(circles) and when antigen is delivered by a continuous pump(triangles).

[0046]FIG. 2 (A and B) are graphs showing the lysis of target cells byCTL versus the effector/target ratio when antigen is delivered as asingle dose (circles), when antigen is delivered by a continuous pump(triangles) and negative control (squares) at (A) 36 hours and (B) 7days.

[0047]FIG. 2C is a graph showing the footpad swelling versus time whenantigen is delivered as a single dose (circles) and when antigen isdelivered by a continuous pump (triangles).

[0048]FIG. 3 is a graph showing the lysis of target cells by CTL versusthe dose of the peptide antigen when the antigen is deliveredsubcutaneously, intravenously and intrasplenically.

[0049]FIG. 4 is a bar graph showing tritiated thymidine uptake in CTLcells induced by antigen introduced intravenously, intrasplenically andsubcutaneously.

[0050]FIG. 5 is a rough schematic of a human lymphatic system.

[0051]FIG. 6. Comparison of anti-peptide CTL responses followingimmunization with various doses of DNA by different routes of injection.

[0052]FIG. 7. Comparison of anamnestic antiviral CTL responses followingimmunization with various doses of DNA by different routes of injection.

[0053]FIG. 8. Protective immunity against systemic and peripheral virusinfection following intra-lymph node immunization with DNA. LCMV titerin spleen (A) and Vacc-G2 vaccinia titers in ovary (B) followingindicated immunization and subsequent viral challenge.

[0054]FIG. 9. Growth of transplanted gp33 expressing tumor in miceimmunized by i.ln. injection of gp33 epitope-expressing, or control,plasmid.

[0055]FIG. 10. Amount of plasmid DNA detected by real-time PCR ininjected or draining lymph nodes at various times after i.ln. of i.m.injection, respectively.

[0056]FIG. 11. Average % supercoiled DNA in formulations 1-9 over 7days.

DETAILED DESCRIPTION OF THE INVENTION Method of Treatment

[0057] One aspect of this invention is a method for inducing orsustaining a specific immunological response (i.e., a CTL response) inan animal that has a disease (or predisposition to a disease) in whichthe animal's immune system may attack the disease with a natural CTLresponse. The response and diseases are discussed in greater detailhereinafter. The method has particular value for treating an animalhaving a malignant tumor in order to inhibit the growth of the tumor orfor treating a chronic infectious disease such as hepatitis or AIDS.

[0058] The method, along with other aspects of the invention, is usefulin an animal having an immune system that includes a lymphatic system.This generally includes vertebrates, specifically mammals andparticularly humans. Thus, this invention will find use in treatinghumans of all ages as well as in treating animals, i.e. in veterinaryuses. The invention may be used for treating livestock such as cattle,sheep, pigs, goats, and the like or for treating household pets such asdogs, cats, rabbits, hamsters, mice, rats, and the like. The primary usewill be for treating humans that are in need of having a specificimmunological response sustained for treatment of a disease such ascancer or chronic infections.

[0059] A key aspect of this invention is the delivery of an appropriateantigen to the lymphatic system of the animal being treated andsustaining the delivery over time. This is based in part on theobservation that a strong induction and a sustained CTL response requireongoing antigenic stimulation of the lymphatic system. In a human, thelymphatic system includes lymph, lymphocytes, lymph vessels, lymphnodes, tonsils, the spleen, the thymus gland, and bone marrow. Thelymphatic system performs three basic functions. First, it helpsmaintain fluid balance in the tissues. Approximately 30 L of fluid passfrom the blood capillaries into the interstitial spaces each day,whereas only 27 L pass from the interstitial spaces back into the bloodcapillaries. If the extra 3 L of interstitial fluid were to remain inthe interstitial spaces, edema would result, causing tissue damage andeventual death. These 3 L of fluid (i.e. lymph) enter the lymphcapillaries, then passes through the lymph vessels to return to theblood. Lymph is similar in composition to plasma. In addition to water,lymph contains solutes derived from two sources: (1) substances inplasma such as ions, nutrients, gases, and some proteins pass from bloodcapillaries into the interstitial spaces to become part of the lymph;and (2) substances derived from cells within the tissues such ashormones, enzymes, and waste products are also found in lymph.

[0060] The lymphatic system's second basic function is to absorb fatsand other substances from the digestive tract. Special lymph vesselscalled lacteals are in the lining of the small intestine. Fats enterinto the lacteals and pass through the lymph vessels to the venouscirculation. The lymph passing through these capillaries has a milkyappearance because of its fat content, and it is called chyle.

[0061] The third basic function of the lymphatic system is to act aspart of the body's defense system. The lymph nodes filter lymph, and thespleen filters blood, removing microorganisms and other foreignsubstances. This third function is the function most important to thisinvention in that the antigen must be delivered to the lymph system at alevel sufficient to elicit the desired, specific immunological responsein the animal. FIG. 5 is a schematic representation of a human lymphaticsystem showing the major lymphatic organs and vessels.

[0062] As hereinbefore mentioned, the present invention relates to amethod of inducing or sustaining a specific immunological response(particularly a CTL response) to an antigen in an animal over time. Themethod comprises delivering the antigen to the animal in a manner thatdelivers the antigen into the lymphatic system of an animal to sustainthe desired response over time. Generally this is done by establishing amechanism to transfer an antigen from a reservoir to the animal'slymphatic system on a regular basis over time. The antigen may bedelivered by a variety of methods that target intralymphaticpresentation, including subcutaneous injection, direct injection intothe lymphatic system by an antigen delivery vehicle that is implanted,preferably at or near a lymphatic organ, or by an antigen deliveryvehicle that is external to the animal but contains a means (e.g. aneedle or catheter) to deliver the antigen into the lymphatic system. Bythis method one can avoid multiple ongoing injections and can also avoidthe use of including professional antigen-presenting cells in thecomposition held in the reservoir.

[0063] The method of this invention can be viewed as inducing CTL immuneresponse by providing high continuous local concentrations of antigen,which otherwise is quickly removed and degraded from the body afterbolus injection. Potent activation of CD8+ T cells requires signalingthrough the T cell receptor (TCR) in a manner that is dependent on bothquantitative and qualitative factors. Quantitative factors refer to thenumber of TCRs engaged by peptide-MHC complexes. Qualitativeconsiderations include the duration of engagement of the TCR bypeptide-MHC complexes, with specific peptide-MFIC complexes. Sustainedregular deliveries of antigen allows optimal conditions to beestablished for inducing CD8+ T cells.

[0064] The antigen is delivered to the animal so that the antigen ispresent in the animal's lymphatic system on a sustained basis over aperiod of time. That is to say, it is delivered in such a way that thepresence of the antigen is maintained over the period of time in theanimal's lymphatic system. Thus, the antigen is delivered to the animalon a regular basis, i.e. the antigen is delivered regularly withoutsignificant interruption over the period of time. This regular deliveryis achieved by the constant delivery of the antigen at low levelsdirectly to the lymphatic system using an external device or animplantable device, as discussed hereinafter. Alternatively, the antigencan be delivered at higher levels to the animal by subcutaneousinjection with indirect absorption or equilibration with the lymphsystem. Delivery on a regular basis is meant to include intermittent(stopping and transmitting at intervals) as well as continuous(transmitting without interruption) delivery. In intermittent delivery,the times transmission is stopped will not be enough to reduce the levelof antigen in the animal's lymphatic system to eliminate the desiredspecific immunological response. Thus, the antigen may be delivered inpulses or small doses over time.

[0065] Preferably, the sustained delivery is achieved by the positioningof a means of delivery so that the animal being treated does not have toreceive multiple injections of the antigen, but instead has only oneinsertion of the means for delivery, e.g. an insertion of a catheter orneedle for infusion of a suitable antigen-containing composition or thesurgical implantation of an implantable device that releases anappropriate, antigen-containing composition on a sustained basis.

[0066] The period of time over which the antigen will be released willbe a time sufficient to induce and maintain the desired specificimmunological response, e.g. to maintain a CTL response, and in the caseof an animal with a tumor or infection, at a level sufficient tostimulate the immune system to attack the tumor and inhibit its growthor to attack the infection. Generally, this period of time may vary froma few days, e.g. a week, to a year or more. Preferably, the treatment,i.e. sustained delivery of the antigen, will extend for at least sevendays and no more than six months. It has been found that the CTLresponse is induced by administration for at least seven days. Todetermine the period of time, the attending physician will evaluate,i.e., the severity of the condition, the strength of the patient, theantigenic response (e.g., the level of CD8+ cells measurable in thepatient's system), the presence of toxic effects, and other factorsknown to one of skill in the art. Ultimately the time for sustaineddelivery in a cancer patient will be that necessary for improvement inthe patient as evidenced by reduction in the size of the tumor, the rateof growth of the tumor, and/or the improvement in the overall health ofthe patient being treated. In the treatment of infectious diseases thetreatment is continued until the health of the patient improvessufficiently to stop treatment.

[0067] The underlying immunological rationale for the utility of thisinvention arises from certain immunological considerations, The immunesystem has evolved to protect the host from microbial infection. CD4+ Tcells together with B cells are the main components of the immune systemhumoral effector arm, which is crucial to eliminate extracellularpathogens or toxins. In contrast, the CD8+ T cell arm of the immunesystem is mainly responsible for eliminating intracellular pathogens,i.e. most importantly viruses, either via cytokine release or bycytotoxic activity. It is now emerging that these most efficient “killercells” of the immune system would best serve as the primary effectorcells in tumor immunotherapy. An object of this invention is to mount adisease-specific CTL response (CD8+ T cell response) against the diseaseand sustain it over time, e.g., a tumor specific or microbial specificCTL response.

[0068] CD8+ T cells recognize antigenic oligopeptides presented on HLAclass I molecules of target cells, e.g., tumor cells. The sequences ofmany HLA-A1 and HLA-A2 presented tumor and pathogen specific antigenpeptides have recently been characterized. These peptides may be used inthis invention to induce, e.g., a melanoma-specific CD8+ T cellresponse. These peptides are discussed hereinafter.

[0069] In contrast to viral infection, class I-binding oligopeptidesshow only low immunogenicity. Most viruses induce peak CD8+ T cellresponses around 7-10 days after systemic spread. This invention aims atenhancing the immunogenecity of class I binding oligopeptides bysustained, regular release of peptide into a lymphatic system andcontinued release into the lymphatic system.

[0070] In contrast to antibody-mediated B cell memory, which is longlived, T cell memory appears to be short lived or non-existent. Inaccordance with this invention, maintenance of functional T cell memorydepends on persistence of antigen through continued, regularadministration of the desired antigen. Having made this invention andlooking at past concepts that might support this underlying rationale,some evidence includes the observation that delayed typehypersensitivity (DTH) of the tuberculin type (the only functional testfor T cell memory in humans), can be elicited only in granulomatousdisease, such as tuberculosis (tuberculin test), leprosy (lepromintest), brucellosis (brucellin test), sarcoidosis (Kveim test),Histoplasmosis (histoplasimin test) etc., but no such test could beestablished for non-granulomatous infectious disease. A factor that allgranulomatous diseases have in common, is that the antigen persistswithin the granuloma-professional antigen presenting cells can use thisreservoir to continuously restimulate specific T cells in lymphoidorgans. In mice models (see Example 3) it is demonstrated thatmaintenance of functional CD8+ T cell memory was strictly dependent oncontinuous antigenic restimulation.

[0071] To determine whether a CTL response is obtained in an animalbeing treated in accordance with this invention, one measures the levelof CD8+ cells (i.e. CTL) present in the blood or lymphatic organs suchas the spleen or lymph nodes. This determination is done by firstmeasuring the level of CD8+ cells before performing the method of thisinvention and measuring the level during treatment, e.g. at 7, 10, 20,40 days, etc. The level or strength of the CD8+ (CTL) response can beassessed in vivo or in vitro. In humans, there exists so far only one invivo test to measure CD8+ T cell responses, which is a skin test. Inthis skin test, HLA class I binding peptides are injected, intradermally(such as described in Jager, E. et al.Granulocyte-macrophage-colony-stimulating Factor Enhances ImmuneResponses To Melanoma-associated Peptides in vivo Int. J Cancer 67,54-62 (1996)). If a CTL response is present, these cells will recognizeand attack peptide pulsed dermal cells, causing a local inflammatoryreaction either via cytokine release or the cytotoxic mechanism (Kündig,T. M., Althage, A., Hengartner, H. & Zinkernagel, R. M. A skin test toassess CD8+ cytotoxic T cell activity. Proc. Natl. Acad Sci. USA89:7757-776 (1992)). This inflammatory reaction can be quantified bymeasuring the diameter of the local skin rash and/or by measuring thediameter of the infiltrate (i.e., the swelling reaction). As analternative to the injection of soluble free peptide, the HLA-class Ibinding peptide can also be injected intradermally in a bound form,e.g., bound to extracorporally derived dendritic cells. In othermammals, additional, although experimental, in vivo tests to assess CD8+T cell responses exist. For example, in a mouse model, CD8+ T cellresponses can be measured by challenge infection with a vacciniarecombinant virus expressing the peptide used for immunization. Whilenaïve mice succumb to the infection with the vaccina recombinant virus,mice with preexisting CD8+ T cell immunity against the peptide epitopeexpressed by the vaccinia recombinant virus, are immune to reinfection.The level of immunity to reinfection can be quantified as the factor ofreduction of the vaccinia virus titer recovered from mouse organs afterchallenge infection (Bachmann, M. F. & Kündig, T. M. In vitro vs. invivo assays for the assessment of T- 655 and B- cell function. Curr.Opin. Immunol. 6, 320-326 (1994)). For example, 5 days after challengeinfection, a typical vaccinia recombinant virus titer recovered from amouse ovary would be around 10⁷ pfu per ovary, whereas the vacciniarecombinant virus titer in a mouse with a preexisting CD8+ T cellresponse against the recombinant gene product would for example bearound 10³ pfu per ovary. Such a 10,000 fold-reduction in virus titerreflects biologically significant preexisting CD8+ T cell activityagainst the recombinant gene product.

[0072] The level of CD8+ T cell responses can also be quantified invitro, by estimating the number of CD8+ T cells specific for theantigenic peptide in question. In a naive mammal the so called“frequency”, i.e., the number of specific CD8+ T cells divided by thenumber of non-specific white blood cells, is less than 10⁻⁶. Aftersuccessful immunization, the frequency increases due to proliferation ofspecific T cells. During an acute viral infection, for example, thefrequency of specific CD8+ T cells may rise to 10⁻². Then, afterelimination of the virus, the frequency of specific CD8+ T cells usuallydrops to a “memory” level of around 10⁻⁴. Thus, the CD8+ T cell responsecan be quantified by measuring the frequency of specific CD8+ T cells.The higher the frequency, the stronger the response. The classicalassays used to measure the frequency of specific CD8+ T cells are basedon limiting dilution cell culture techniques, as described in detail byKündig, T. M. et al. (On the role of antigen in maintaining cytotoxic Tcell memory. Proceedings of the National Academy of Sciences of theUnited States of America 93, 9716 972′) (1996)). A novel approach toestimate the frequency of specific CD8+ T cells is to construct solubleclass I MHC (for use in mice) or HLA molecules (for use in humans) witha peptide bound to their groove, so that the specific T cell receptorswill bind to these complexes. These complexes can be labeled fordetection, for example, with a fluorescent substance, allowing fordetection by flow cytometry.

[0073] One current procedure to render peptides immunogenic is to injectthem in context with “nature's most potent adjuvant”, i.e., professionalantigen presenting cells (APCs) such as dendritic cells (DCs),(Steinmann, R. M., The dendritic cells system and its role inirnmunogenicity, Annual Review of Immunology 9, 271-96 (1991)). DCs arethe most potent APCs of the immune system. They can now be cultured invitro by adding granulocyte macrophage colony stimulating factor(GM-CSF) and tumor necrosis factor alpha (TNF-alpha) or interleukin-4(IL-4) to progenitors isolated from the blood of patients or mice(Inaba, K. el al., Identification of proliferating dendritic cellprecursors in mouse blood, Journal of Experimental Medicine 175,1157-1167 (1992)). Large numbers of DCs can then be pulsed with tumorspecific antigen peptides and are injected back into the patient, wherethey migrate into lymphatic organs to induce T cell responses (Young, J.W. & Inaba, K., Dendritic Cells As Adjuvants For Class I MajorHistocompatibility Complex-restricted Anti-tumor Immunity, Journal ofExperimental Medicine 183, 7-11 (1996)). An object of this invention isto circumvent the time-consuming, labor intensive procedure of culturingDCs after isolation of DC progenitors and deliver the antigen to thelymphatic system free of APCs such as DCs. The method of this invention,i.e., the sustained, regular delivery of antigen into a lymphatic organ,allows sufficiently high local concentrations of antigen inside thelymphatic organ, such that professional antigen presenting cells, e.g.,dendritic cells, can be loaded with peptide in vivo. This can be viewedas a method of loading antigen presenting cells (dendritic cells) invivo for inducing a CTL response.

[0074] The method of the present invention is clearly advantageous overthe prior art methods for inducing a CTL response against a tumor orvirus. For example, the present invention does not require repetitiveimmunizations to effect for prolonged anti-tumor immunotherapy. Thesustained delivery of the antigen maintains the CTL response that couldultimately afford a prolonged aggressive posture of CTL against tumorcells, more thorough eradication, and protection against recurrenceduring the vaccine treatment. In the absence of antigen, CTL that haveundergone primary activation soon cease to recirculate through the body,soon finding their way to the spleen where they become quiescent. SinceCTL must immediately deliver a lethal hit, their residence in the spleenprecludes an active role in protection against infections or tumorgrowth at distant sites in the body. The controlled release of antigenrecognized by CTL in this invention circumvents this outcome as antigendelivery is maintained. Sustained released antigen delivery to thelymphatic system by this invention solves two major problems: itprovides for potent CTL stimulation that takes place in the milieu ofthe lymphoid organ, and it sustains stimulation that is necessary tokeep CTL active, cytotoxic and recirculating through the body.

[0075] Another fundamental improvement of the present method over priorart is that it facilitates the use of inherently non-immunogenic peptideantigens for CTL stimulation without the combined use of conventionaladjuvants. This is very beneficial as most experimental adjuvants aretoxic and poorly suited for use in humans. In addition adjuvantsstimulate the TH2-type humoral immune response that negatively affectsthe CTL response. Further, since conventional adjuvants are notrequired, only the minimal antigenic epitope for a CTL response isrequired in the formulation.

[0076] An additional advantage to the method of the present invention,where it embodies the use of mechanical delivery systems, is that theantigen delivery can be stopped if any adverse immunological effects areobserved, For example, in vaccines against melanoma, CTL have beeninduced to attack not only malignant melanocytes but also healthytissue, causing “vitiligo.” The ability to discontinue a CTL vaccine atany time is a significant advance in vaccine safety. Peptides have ashort half-life due to catabolism in the liver. Therefore, thestimulation-effect falls soon after cessation of delivery.

[0077] As pointed out before, the method of this invention has twoparts: (1) inducing an increased CTL response and (2) maintaining theresponse. The inducing and maintaining may be performed using the samedevice, as discussed hereinafter, or the inducing may be doneseparately, e.g., by a separate injection of an antigen then followingup with sustained delivery of the antigen over time to maintain theresponse.

Diseases Treated According to the Invention

[0078] In general, this invention is useful for treating an animalhaving (or being predisposed to) any disease to which the animal'simmune system mounts a cell-mediated response to a disease-relatedantigen in order to attack the disease. Thus, the type of disease may bea malignant tumor or a chronic infectious disease caused by a bacterium,virus, protozoan, helminth, or other microbial pathogen that entersintracellularly and is attacked, i.e., by the cytotoxic T lymphocytes,In addition, the invention is useful for treating an animal that may beat risk of developing such diseases.

Malignant Tumors

[0079] In a mature animal, a balance usually is maintained between cellrenewal and cell death in most organs and tissues. The various types ofmature cells in the body have a given life span; as these cells die, newcells are generated by the proliferation and differentiation of varioustypes of stem cells. Under normal circumstances, the production of newcells is so regulated that the numbers of any particular type of cellremain constant. Occasionally, though, cells arise that are no longerresponsive to normal growth-control mechanisms. These cells give rise toclones of cells that can expand to a considerable size, producing atumor, or neoplasm. A tumor that is not capable of indefinite growth anddoes not invade the healthy surrounding tissue extensively is benign. Atumor that continues to grow and becomes progressively invasive ismalignant; the term cancer refers specifically to a malignant tumor. Inaddition to uncontrolled growth, malignant tumors exhibit metastasis; inthis process, small clusters of cancerous cells dislodge from a tumor,invade the blood or lymphatic vessels, and are carried to other tissues,where they continue to proliferate. In this way a primary tumor at onesite can give rise to a secondary tumor at another site. The methods,devices and articles of manufacture discussed herein are useful fortreating animals having malignant tumors.

[0080] Malignant tumors treated according to this invention areclassified according to the embryonic origin of the tissue from whichthe tumor is derived. Carcinomas are tumors arising from endodermal orectodermal tissues such as skin or the epithelial lining of internalorgans and glands. A melanoma is a type of carcinoma of the skin forwhich this invention is particularly useful. Sarcomas, which arise lessfrequently, are derived from mesodermal connective tissues such as bone,fat, and cartilage. The leukemias and lymphomas are malignant tumors ofhematopoietic cells of the bone marrow. Leukemias proliferate as singlecells, whereas lymphomas tend to grow as tumor masses. The malignanttumors may show up at numerous organs or tissues of the body toestablish a cancer. The types of cancer that can be treated inaccordance with this invention include the following: bladder, brain,breast, cervical, colo-rectal, esophageal, kidney, liver, lung,nasopharangeal, pancreatic, prostate, skin, stomach, uterine, and thelike. The present invention is not limited to the treatment of anexisting tumor or infectious disease but can also be used to prevent orlower the risk of developing such diseases in an individual, ie., forprophylactic use. Potential candidates for prophylactic vaccinationinclude individuals with a high risk of developing cancer, i.e., with apersonal or tuminal history of certain types of cancer.

[0081] The incidence of skin cancer has increased substantially over thelast decades. Lifetime analysis indicates that around 1/1500 humans bornin 1935, 1/600 born in 1960, 1/100 born in 1990 and a projected 1/75humans born in the year 2000 will have melanoma in their lifetime.Surgical excision usually cures melanoma. However, even small lookinglesions may have already metastasized at the time of diagnosis. Theprognosis of metastasized melanoma is very poor and correlates with thethickness of the primary tumor and with its localization.

[0082] The current treatment of malignant melanoma aims at surgicalremoval of the primary tumor, If metastases are present, chemotherapyand biological response modifiers are additionally used. However,patients with stage IV malignant melanoma are almost invariablyincurable and treatments are palliative. Patients with Stage IVmalignant melanoma have a median survival time of approximately one yearand only a 10% chance of long-term survival. There is at present nogenerally accepted standard therapy for metastatic melanoma. Objectiveresponse rates to mono- or polychemotherapy are low in comparison withother tumors, reaching no more than 15-35%. An improved treatmentoutcome in stage IV malignant melanoma seems unachievable either bychemotherapeutic combinations or by increasing doses to levels whereautologous bone marrow transplantation becomes necessary. The method ofthis invention is useful for treating malignant melanoma, even at StageIV.

Infectious Diseases

[0083] Infectious diseases, which have plagued animal populations(particularly humans) throughout history, still cause millions of deathseach year. The infectious diseases that can be treated using thisinvention include those caused by pathogens such as bacteria, viruses,protozoa, helminths, and the like. These diseases include such chronicdiseases such as acute respiratory infections, diarrheal diseases,tuberculosis, malaria, hepatitis (hepatitis A, B C, D, E, F virus),measles, mononucleosis (Epstein-Barr virus), whooping cough (pertussis),AIDS (human immunodeficiency virus I & 2), rabies, yellow fever, and thelike. Other diseases caused by human papilloma virus or various strainsof virus are treatable by this method.

[0084] In some instances, the mammal, in particular human, can betreated prophylactically, such as when there may be a risk of developingdisease. An individual travelling to or living in an area of endemicinfectious disease may be considered to be at risk and a candidate forprophylactic vaccination against the particular infectious agent. Forexample, the CTL response can be induced in a human expecting to enter amalarial area and/or while in the malarial area by using a CTL-inducing,-malaria-specific antigen to lower the risk of developing malaria.Preventative treatment can be applied to any number of diseasesincluding those listed above, where there is a known relationshipbetween the particular disease and a particular risk factor, such asgeographical location or work environment.

Antigens Useful in the Invention

[0085] An antigen useful in this invention is one that stimulates theimmune system of a mammal having a malignant tumor or infectious diseaseto attack the tumor and inhibit its growth or to destroy the pathogencausing the disease. Thus, the antigen used in the invention is matchedto the specific disease found in the animal being treated. In thisregard the antigen may be said to induce a CTL response (also referredto as a cell-mediated immune response), i.e. a cytotoxic reaction by theimmune system that results in lysis of the target cells (e.g., themalignant tumor cells or pathogen-infected cells).

[0086] To determine whether an antigen is matched to a particularpatient, whether human or other animal, the tissue type of the patientis first determined. If human, the tissue must demonstrate theappropriate human leukocyte antigen (HLA) capable of binding anddisplaying the antigen to CTL. It is preferable that the HLA typing beperformed, on the target cells, since a significant portion of tumorsescape immune detection by downregulating expression of HLA. ThereforeHLA expression on normal cells of the patient does not necessarilyreflect that found on tumor cells in their body. A tumor from a patientis also screened to determine if he or she expresses the antigen that isbeing used in the vaccine formulation. Immunohistochemistry and/orpolymerase chain reaction (PCR) techniques both can be used to detectantigen in the tumor cells. Immunchistochemistry offers the advantage inthat it stains a cross-section of tumor in a slide preparation, allowinginvestigators to observe the antigen expression pattern in cross-sectionof tumor, which is typically heterogeneous for antigen expression. PCRhas the advantage of not requiring specific monoclonal antibodies forstaining and is a fast and powerful technique. In addition, PCR can beapplied in situ. Ideally, both immunohistochemical and PCR methodsshould be combined when assessing antigen expression in tumors. Whilethe antigen compositions useful in this invention are designed toinclude the most commonly expressed tumor antigens (as discussedhereafter), not all tumors will express the desired antigen(s). Where atumor fails to express the desired antigen, the patient is excluded forconsideration for that particular antigen composition. Thus, an aspectof this invention is a process for preparing a device useful forproviding a sustained CTL response over time by matching a subject'santigen specific to the tumor or pathogen in the subject, preparing aphysiologically-acceptable composition of the antigen so matched, andcombining the composition in a suitable delivery device as discussed inhereinafter.

[0087] Immune activation of CD8+ T cells generates a population ofeffector cells with lytic capability called cytotoxic T lymphocytes, orCTL. These effector cells have important roles in the recognition andelimination of malignant cells and pathogens. In general, CTL are CD8+and are therefore class I MHC restricted, although in rare instancesCD4+ class II-restricted T cells have been shown to function as CTL.Since virtually all nucleated cells in the body express class I MHCmolecules, CTL can recognize and eliminate almost any altered body cell.CD8+ T cells recognize antigen presented on HLA class I molecules oftumor cells through T cell receptors.

[0088] The CTL-mediated immune response can be divided into two phases,reflecting different aspects of the cytotoxic T-cell response. The firstphase involves the activation and differentiation of T_(c) (CD8+) cellsinto functional effector CTLs. In the second phase, CTLs, recognizeantigen-class I MHC complexes on specific target cells, initiating asequence of events that culminates in target-cell destruction. Furtherdetailed discussion of the process is found at Chapter 15 of the SecondEdition of “Immunology” by Janis Kuby, W. H. Freeman and Company (1991).

[0089] The type of tumor antigen used in this invention may be atumor-specific antigen (TSA) or a tumor-associated antigen (TAA), A TSAis unique to tumor cells and does not occur on other cells in the body.A TAA associated antigen is not unique to a tumor cell and instead isalso expressed on a normal cell under conditions that fail to induce astate of immunologic tolerance to the antigen. The expression of theantigen on the tumor may occur under conditions that enable the immunesystem to respond to the antigen. TAAs may be antigens that areexpressed on normal cells during fetal development when the immunesystem is immature and unable to respond or they may be antigens thatare normally present at extremely low levels on normal cells but whichare expressed at much higher levels on tumor cells. TSAs and TAAs can bejointly referred to as TRA or a tumor related antigen.

[0090] Tumor antigens useful in the present invention, whethertumor-specific or tumor-associated, must be capable of inducing aCTL-mediated immune response. The presence of tumor antigens that elicita cell-mediated response has been demonstrated by the rejection oftumors transplanted into syngeneic recipients; because of thisphenomenon, these tumor antigens are referred to as tumor-specifictransplantation antigens (TSTAs) or tumor-associated transplantationantigens (TATAs). It has been difficult to characterize tumortransplantation antigens because they do not generally elicit anantibody response and therefore they cannot be isolated byimmunoprecipitation. Many are peptides that are presented together withMHC molecules on the surface of tumor cells and have been characterizedby their ability to induce an antigen-specific CTL.

[0091] The type of pathogen specific antigen useful in this inventionmay be short oligopeptides derived from pathogen proteins. Theseoligopeptides must bind to class I MHC (for use in mice), class I HLA(for use in humans), or class I molecules of any other mammals. Also,such class I molecule bound peptides should be recognizable by specificT cell receptors. Such oligopeptides usually have a length of 8-15 aminoacids. Several examples of such pathogen derived oligopeptides, socalled T cell epitopes, are given in Tables I and II.

[0092] The tumor antigens and pathogen-specific antigens useful in thisinvention are generally thought to be presented at the surface of anantigen presenting cell (APC) to stimulate the immune system throughclass I molecules of the major histocompatability complex (MHC)interactively with the CD8+ cells.

[0093] Antigens useful in the invention are generally protein-basedentities of a molecular weight of up to 100,000 daltons. Appropriateantigens include, but are not limited to differentiation antigens,tumor-specific multilineage antigens, embryonic antigens, antigens ofoncogenes and mutated tumor-suppressor genes, unique tumor antigensresulting from chromosomal translocations, viral antigens, and othersthat may be apparent presently or in the future to one of skill in theart. It is preferable that the antigen be a peptide of 8 to 15 aminoacids in length that is an epitope of a larger antigen, i.e. it is apeptide having an amino acid sequence corresponding to the site on thelarger molecule that is recognized and bound by a particular T-cellreceptor. These smaller peptides are available to one of skill in theart by following the teachings of U.S. Pat. No. 5,747,269 to Rarnmenseeet al. issued May 5, 1998; U.S. Pat. No. 5,698,396 to Pfreundschuhissued Dec. 16, 1997; and PCT Application Numbers PCT/EP95/02593 filedJul. 4, 1995, PCT/DE96/00351 filed Feb. 26, 1996, all of which areincorporated herein by reference. Additional approaches to epitopediscovery are described in U.S. Pat. No. 6,037,135 METHODS FOR MAKINGHLA BINDING PEPTIDES AND THEIR USES and U.S. patent application Ser. No.09/561,074 entitled METHOD OF EPITOPE DISCOVERY both of which areincorporated herein by reference in their entirety.

[0094] While in the general case the antigen ultimately recognized by aT cell is a peptide, it must be kept in mind that the form of antigenactually administered as the immunogenic preparation need not be apeptide per se. When administered, the epitopic peptide(s) may residewithin a longer polypeptide, whether the complete protein antigen, somesegment of it, or some engineered sequence. Included in such engineeredsequences would be polyepitopes and epitopes incorporated into somecarrier sequence such as an antibody or viral capsid protein. Suchlonger polypeptides may include epitope clusters as described in U.S.patent application Ser. No. 09/561,571 entitled “EPITOPE CLUSTERS,”which is incorporated herein by reference in its entirety. The epitopicpeptide, or the longer polypeptide in which it is contained, may be acomponent of a microorganism (e.g. a virus, bacterium, protozoan, etc.),or a mammalian cell (e.g. a tumor cell or antigen presenting cell), orlysates, whole or partially purified, of any of the foregoing. They maybe used as complexes with other proteins, for example heat shockproteins. The epitopic peptide may also be covalently modified, such asby lipidation, or made a component of a synthetic compound, such asdendrimers, multiple antigen peptides systems (MAPS), and polyoximes, ormay be incorporated into liposomes or microshperes, etc. As used in thisdisclosure the term “polypeptide antigen” encompasses all suchpossibilities and combinations. The invention comprehends that theantigen may be a native component of the microorganism or mammaliancell. The antigen may also be expressed by the microorganism ormammalian cell through recombinant DNA technology or, especially in thecase of antigen presenting cells, by pulsing the cell with polypeptideantigen prior to administration. Additionally, the antigen may beadministered encoded by a nucleic acid that is subsequently expressed byAPCs. Finally, whereas the classical class I MHC molecules presentpeptide antigens, there are additional class I molecules which areadapted to present non-peptide macromolecules, particularly componentsof microbial cell walls, including without limitation lipids andglycolipids. As used in this disclosure the term antigen comprehendssuch macromolecules as well. Moreover, a nucleic acid based vaccine mayencode an enzyme or enzymes necessary to the synthesis of such amacromolecule and thereby confer antigen expression on an APC.

[0095] A powerful method has been recently developed for identifying newpeptides that are useful in the invention. Genes determined to expressprotein with high exclusivity in tumor cells or microbial cells (e.g.viruses) can be identified using a so called SEREX process, whichinvolves expression cloning using tumor cell libraries and screeningthese libraries against immunoglobulin in patient sera. Over one hundredgenes have recently been identified from tumor biopsies using thisprocess. These genes can now be used in a peptide prediction algorithmdeveloped by Hans-Georg Rammensee. Algorithms have been developed forall major HLA types found in the human population. First the proteinsequence is “translated” based on the gene sequence. The algorithms canpredict peptide epitopes for various HLA types based on the proteinsequence. Since the predicted peptides are indeed predictions and arenot always naturally found on cells, tumor samples are used to confirmthe predicted peptides by actually isolating minute trace peptide fromtumors. Being able to calculate the exact mass of the predicted peptidesallows trace peptide identification using ultrasensitive massspectrophotometry, which can detect peptides in quantities less thatthat which would permit peptide sequencing and identification. Oncethese tumor-associated peptides have been identified they are suitablefor use in the invention, since peptides of a known sequence may besynthesized in large quantities (several grams) providing for sufficientamounts of peptides for use in this invention.

[0096] In addition to the imperfection of existing prediction algorithmsfor MHC binding, some peptides that would be fully capable of binding toMHC may never be liberated by protoelytic processing from the completeprotein antigen. Methods for evaluating which peptides will be liberatedby proteasomal processing have been developed, e.g. U.S. patentapplication Ser. No. 09/561,074 supra, increasing the efficiency withwhich useful epitopes can be discovered. Moreover, proteasomalprocessing can differ between target cell and APC such that care must betaken in the identification and selection of epitopes and in vaccinedesign so that the vaccine will induce a response that will in factrecognize the target cell. These issues are more fully discussed in U.S.patent application Ser. No. 09/560,465 entitled “EPITOPESYNCHRONIZATION,” which is incorporated herein by reference in itsentirety.

[0097] Thus it can be seen that another aspect of this invention is aprocess for preparing a composition useful in a device of this inventionas discussed hereinafter. The process comprises identifying a genedetermined to express a protein with high exclusivity in a tumor ormicrobial cell, cloning cell libraries, screening the libraries againstimmunoglobulin in patient sera, using the algorithm defined in theliterature developed by Hans-George Rammensee to predict an epitope forthe HLA type protein based on the gene sequence, matching the predictedantigen sequence to a patient tumor sample, isolating the matchedantigen, and preparing a composition of the antigen for use in adelivery device as discussed hereinafter.

[0098] Examples of large, protein-based antigens include the following:

[0099] Differentiation antigens such as MART-1/MelanA (MART-I), gp100(Pmel 17), tyrosinase, TRP-1, TRP-2 and tumor-specific multilineageantigens such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, p15;overexpressed embryonic antigens such as CEA; overexpressed oncogenesand mutated tumor-suppressor genes such as p53, Ras, HER-2/neu; uniquetumor antigens resulting from chromosomal translocations; such asBCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR; and viral antigens, such asthe Epstein Barr virus antigens EBVA and the human papillomavirus (HPV)antigens E6 and E7. Other large, protein-based antigens include TSP-180,MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, p185erbB2, p180erbB-3, c-met,nm-23HI, PSA, TAG-72, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras,β-Catenin, CDK4, Mum-1, p 15, p 16, 43-9F, 5T4, 791Tgp72,alpha-fetoprotein , β-HCG, BCA225, BTAA, CA 125, CA 15-3CA \27.29\BCAA,CA 195, CA 242, CA-50, CAM43, CD68\KP1, CO-029, FGF-5, G250,Ga733\EpCAM, HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB/70K, NY-CO-1,RCAS1, SDCCAG16, TA-90\Mac-2 binding protein\cyclophilin C-associatedprotein, TAAL6, TAG72, TLP, and TPS. These protein-based antigens areknown and available to those of skill in the art in the literature orcommercially.

[0100] Examples of peptide antigens of 8-15 amino acids include thoseset forth in Table I, Table II, and Table III. Table I sets forthantigens that are virally derived. The Table shows the virus type, theprotein expressed by the virus, the amino acid (AA) position on theviral protein, the AA sequence of the T-cell epitope/MHC ligand, thetype of MHC molecule presenting the antigen, and a reference source. Amore complete list is provided in the book by Han-Georg Rammensee, JuttaBachmann, and Stefan Stevanovic entitled “MHC Ligands and PeptideMotifs,” Springer-Verlag, Germany, 1997 Landes Bioscience, Austin,Tex.). The reference number given in Table I is the same number (andreference source) given in Table 5.3 of the above Rammensee book, all ofwhich is incorporated herein by reference. TABLE I Viral epitopes on MHCclass 1 molecules T cell epitope MHC Virus Protein AA Position ligand(Antigen) MHC molecule Ref. Adenovirus 3 E3 9Kd 30-38 LIVIGILILHLA-A*0201 104 (SEQ. ID NO.:1) Adenovirus 5 EIA 234-243 SGPSNTPPEI H2-Db105 (SEQ. ID NO.:2) Adenovirus 5 EIB 192-200 VNIRNCCYI H2-Db 106 (SEQ.ID NO.:3) Adenovirus 5 EIA 234-243 SGPSNIPPEI (T > I) H2-Db 106 (SEQ. IDNO.:4) CSFV NS 2276-2284 ENALLVALF SLA, haplotype 107 polyprotein (SEQ.ID NO.:5 d/d Dengue virus 4 NS 3500-508 TPEGIIPTL HLA-B*3501 108, 109(SEQ. ID NO.:6 EBV LMP-2 426-434 CLGGLLTMV HLA-A*0201 110 (SEQ. IDNO.:7) EBV EBNA-1 480-484 NIAEGLRAL HLA-A*0201 111 (SEQ. ID NO.:8) EBVEBNA-1 519-527 NLRRGTALA HLA-A*0201 111 (SEQ. ID NO.:9) EBV EBNA-1525-533 ALAIPQCRL HLA-A*0201 111 (SEQ. ID NO.:10) EBV EBNA-1 575-582VLKDAIKDL HLA-A*0201 111 (SEQ. ID NO.:11) EBV EBNA-1 562-570 FMVFLQTHIHLA-A*0201 111 (SEQ. ID NO.:12) EBV EBNA-2 15-23 HLIVDTDSL HLA-A*0201111 (SEQ. ID NO.:13) EBV EBNA-2 22-30 SLGNPSLSV HLA-A*0201 111 (SEQ. IDNO.:14) EBV EBNA-2 126-134 PLASAMRML HLA-A*0201 111 (SEQ. ID NO.:15) EBVEBNA-2 132-140 RMLWMANY1 HLA-A*0201 111 (SEQ.ID NO.:16) EBV EBNA-2133-141 MLWMANYIV HLA-A*0201 111 (SEQ. ID NO.:17) EBV EBNA-2 151-159ILPQGPQTA HLA-A*0201 111 (SEQ. ID NO.:18) EBV EBNA-2 171-179 PLRPTAPTIHLA-A*0201 111 (SEQ. ID NO.:19) EBV EBNA-2 205-213 PLPPATLTV HLA-A*0201111 (SEQ. ID NO.:20) EBV EBNA-2 246-254 RMHLPVLHV HLA-A* 0201 111 (SEQ.ID NO.:21) EBV EBNA-2 287-295 PMPLPPSQL HLA-A*0201 111 (SEQ. ID NO.:22)EBV EBNA-2 294-302 QLPPPAAPA HLA-A*0201 111 (SEQ. ID NO.:23) EBV EBNA-2381-389 SMPELSPVL HLA-A*0201 111 (SEQ. ID NO.:24) EBV EBNA-2 453-461DLDESWDYI HLA-A*0201 111 (SEQ. ID NO.:25) EBV BZLF1 43-51 PLPCVLWPVHLA-A*0201 111 (SEQ. ID NO.:26) EBV BZLF1 167-175 SLEECDSEL HLA-A*02O1111 (SEQ. ID NO..27) EBV BZLF1 176-184 EIKRYKNRV HLA-A*0201 111 (SEQ. IDNO.:28) EBV BZLF1 195-203 QLLQHYREV HLA-*0201 111 (SEQ. ID NO.:29) EBVBZLF1 196-204 LLQHYREVA HLA-A*0201 111 (SEQ. ID NO.:30) EBV BZLFI217-225 LLKQMCPSL HLA-A*O201 111 (SEQ. ID NO.:31) EBV BZLF1 229-237SIIPRTPDV HLA-A*0201 111 (SEQ. ID NO.:32) EBV EBNA-6 284-293 LLDFVRFMGVHLA-A*0201 112 (SEQ. ID NO.:33) EBV EBNA-3 464-472 SVRDRLARL HLA-A*0203113 (SEQ. ID NO.:34) EBV EBNA-4 416-424 IVTDFSVIK HLA-*1101 114,115(SEQ. ID NO.:35) EBV EBNA-4 399-408 AVFDRKSDAK HLA-A*0201 116 (SEQ. IDNO.:36) EBV EBNA-3 246-253 RYSIFFDY HLA-A24 113 (SEQ. ID NO.:37) EBVEBNA-6 881-889 QPRAPIRPI HLA-B7 117 (SEQ. ID NO.:38) EBV EBNA-3 379-387RPPIFIRRI. HLA-B7 117 (SEQ. ID NO.:39) EBV EBNA-1 426-434 EPDVPPGAIHLA-B7 111 (SEQ. ID NO.:40) EBV EBNA-1 228-236 IPQCRLTPL HLA-B7 111(SEQ. ID NO.:41) EBV EBNA-1 546-554 GPGPQPGPL HLA-B7 111 (SEQ. IDNO.:42) EBV EBNA-1 550-558 QPGPLRESI HLA-B7 111 (SEQ. ID NO.:43) EBVEBNA-1 72-80 R.PQKR1~SCI HLA-B7 111 (SEQ. ID NO.:44) EBV EBNA-2 224-232PPTPLLTVL HLA-B7 111 (SEQ. ID NO.:45) EBV EBNA-2 241-249 TPSPPRMHLHLA-B7 111 (SEQ. ID NO.:46) EBV EBNA-2 244-252 PPRMHLPVL HLA-B7 111(SEQ. ID NO.:47) EBV EBNA-2 254-262 VPDQSMHPL HLA-B7 111 (SEQ. IDNO.:48) EBV EBNA-2 446-454 PPSIDPADL HLA-B7 111 (SEQ. ID NO.:49) EBVBZLFI 44-52 LPCVLWPVL HLA-B7 111 (SEQ. ID NO.:50) EBV BZLF1 222-231CPSLDVDSII HLA-B7 111 (SEQ. ID NO.:51) EBV BZLFI 234-242 TPDVLHEDLHLA-B7 111 (SEQ. ID NO.:52) EBV EBNA-3 339-347 FLRGRAYGL HLA-B8 118(SEQ. ID NO.:53) EBV EBNA-3 26-34 QAKWRLQTL HLA-B8 113 (SEQ. ID NO.:54)EBV EBNA-3 325-333 AYPLHEQHG HLA-B8 116 (SEQ. ID NO.:55) EBV EBNA-3158-166 Y1KSFVSDA HLA-B8 116 (SEQ. ID NO.:56) EBV LMP-2 236-244RRRWRRLTV HLA-B*2704 119 (SEQ. ID NO.:57) EBV EBNA-6 258-266 RRJYDLIELHLA-B*2705 119 (SEQ. ID NO.:58) EBV EBNA-3 458-466 YPLHEQHGM HLA-B*3501120 (SEQ. ID NO.:59) EBV EBNA-3 458-466 YPLHEQHGM HLA-B*35O3 113 (SEQ.ID NO.:59) HCV NS3 389-397 HSKKKCDEL HLA-B8 145 (SEQ. ID NO.:60) HCV envE 44-51 ASRCWVAM HLA-B*3501 146 (SEQ. ID NO.:61) HCV core protein 27-35GQIVGGVYL HLA-B*40012 147 (SEQ. ID NO.:62) HCV NSI 77-85 PPLTDFDQGWHLA-B*5301 145 (SEQ. ID NO.:63) HCV core protein 18-27 LMGYIPLVGA H2-Dd138 (SEQ. ID NO.:64) HCV/core protein 16-25 ADLMGYIPLV H2-Dd 148 (SEQ.ID NO.:65) HCV NS5 409-424 MSYSWTGALVTPC H2-Dd 149 AEE (SEQ. ID NO.:66)HGV NSI 205-213 KHPDATYSR Papa-A06 150 (SEQ. ID NO.:67) HCV-1 NS3400-409 KLVALG17NAV HLA~A*0201 141 (SEQ. ID NO.:68) HCV-1 NS3 440-448GDFDSVIDC Patr-B16 151 (SEQ. ID NO.:69) HCV-1 env E 118-126 GNASRCWVAPatr-B16 151 (SEQ. ID NO.:70) HCV-1 NSI 159-167 TRPPLGNWF Patr-B13 151(SEQ. ID NO.:71) HCV-1 NS3 351-359 VPHPNIEEV Patr-B13 151 (SEQ. IDNO.:72) HCV-1 NS3 438-446 YTGDFDSVI Patr-B01 151 (SEQ. ID NO.:73) HCV-1NS4 328-335 SWAIKWEY Patr-A11 151 (SEQ. ID NO.:74) HGV-1 NSI 205-213KHPDATYSR Patr-A04 150 (SEQ. ID NO.:75) HCV-1 NS3 440-448 GDFDSVIDCPatr-A04 150 (SEQ. ID NO.:76) HIV gp4 1583-591 RYLKDQQLL HLA——A24 152(SEQ. ID NO.:77) HIV gagp24 267-275 IVGLNKIVR HLA-A*3302 153, 154 (SEQ.ID NO.:78) HIV gagp24 262-270 EIYKRWIIL HLA-B8 155, 156 (SEQ. ID NO.:79)HIV gagp24 261-269 GE1YKRWIL HLA-B8 155, 156 (SEQ. ID NO.:80) HIV gagp1793-101 EIKDTKEAL HLA-B8 155, 157 (SEQ. ID NO.:81) HIV gp41 586-593YLKDQQLL HLA-B8 158 (SEQ. ID NO.:82) HIV gagp24 267-277 ILGLNK1YRMYHLA-B*1501 153 (SEQ. ID NO.:83) HIV gp41 584-592 ERYLKDQQL HLA-B14 158(SEQ. ID NO.:84) HIV nef 115--125 YHTQGYFPQWQ HLA-B17 159 (SEQ. IDNO.:85) HIV nef 117-128 TQGYFPQWQNYT HLA-817 159 (SEQ. ID NO.:86) HIVgp120 314-322 GRAFVT1GK HLA-B*2705 160, 184 (SEQ. ID NO.:87) HIV gagp24263-271 KRWIILGLN HLA-B*27O2 161 (SEQ. ID NO.:88) HIV nef 72-82QVPLRPMTYK HLA-B*3501 159 (SEQ. ID NO.:89) HIV nef 117-125 TQGYFPQWQHLA-B*3701 159 (SEQ. ID NO.:90) HIV gagp24 143-151 HQAISPRTI,HLA-Cw*0301 162 (SEQ. ID NO.:91) HIV gagp24 140-15 1 QMVHQAISPRTLHLA-Cw*0301 162 (SEQ. ID NO.:92) HIV gp120 431-440 MYAPPIGGQI H2-Kd 163(SEQ. ID NO.:93) HIV gp160 318-327 RGPGRAFVTI H2-Dd 164, 165 (SEQ. IDNO.:94) HIV gp120 17-29 MPGRAFVTI H2-Ld 166, 167 (SEQ. ID NO.:95) HIV-1RT 476-484 ILKEPVIIGV HLA-A*0201 168, 169 (SEQ. ID NO.:96) HIV-1 nef190-198 AFHHVAREL HLA-A*0201 170 (SEQ. ID NO.:97) HIV-1 gpI60 120-128KLTPLCVTL HLA-A*0201 171 (SEQ. ID NO.:98) HIV-1 gp]60 814-823 SLLNATDIAVHLA-A*0201 171 (SEQ. ID NO.:99) HIV-1 RT 179--87 VIYQYMDDL HLA-A*0201172 (SEQ.ID NO.:100) HIV-1 gag p 17 77-85 SLYNTVATL HLA-A*0201 173(SEQ.ID NO.:101) HIV-1 gp160 315-329 RGPGRAFVT1 HLA-A*0201 174 (SEQ. IDNO.:102) HIV-1 gp41 768-778 RLRDLLLIVTR HLA-A3 175, 178 (SEQ. IDNO.:103) HIV-1 nef 73-82 QVPLRPMTYK HLA-A3 176 (SEQ. ID NO.:104) HIV-1gp120 36-45 TVYYGVPVWK HLA-A3 177 (SEQ. ID NO.:105) HIV-1 gagp 17 20-29RLRPGGKKK HLA-A3 177 (SEQ. ID NO.:106) HIV-1 gp120 38-46 VYYGVPVWKHLA-A3 179 (SEQ. ID NO.:107) HIV-1 nef 74-82 VPLRPMTYK HLA-a*1101 114(SEQ. ID NO.:108) HIV-1 gagp24 325-333 AIFQSSMTK HLA-A*1101 114 (SEQ. IDNO.:109) HIV-1 nef 73-82 QVPLRPMTYK HLA-A*1101 180 (SEQ. ID NO.:104)HIV-1 nef 83-94 AAVDLSHFLKEK HLA-A*1101 159 (SEQ. ID NO.:110) HIV-1gagp24 349-359 ACQGVGGPGGHK HLA-A*1101 181 (SEQ. ID NO.:111) HIV-1gagp24 203-212 ETINEEAAEW HLA-A25 182 (SEQ. ID NO.:112) HIV-1 nef128-137 TPGPGVRYPL HLA-B7 159 (SEQ. ID NO.:113) HIV-1 gagp 17 24-31GGKKKYKL HLA-B8 183 (SEQ. ID NO.:114) HIV-1 gp120 2-10 RVKEKYQHL HLA-B8181 (SEQ. ID NO.:115) HIV-1 gagp24 298-306 DRFYKTLRA HLA-B14 173 (SEQ.ID NO.:116) HIV-1 NEF 132-147 GVRYPLTFGWGYKL HLA-B18 159 VP (SEQ. IDNO.:117) HIV-1 gagp24 265-24 KRWIILGLNK HLA-B*2705 184, 153 (SEQ. IDNO.:118) HIV-1 nef 190-198 AFHHVAREL HLA-B*5201 170 (SEQ. ID NO.:97) EBVEBNA-6 335-343 KEIIVIQNAF HLA-B44 121 (SEQ. ID NO.:119) EBV EBNA-6130-139 EENLLDFVRF HLA-B*4403 122 (SEQ. ID NO.:120) EBV EBNA-2 42-51DTPLLPLTIF HLA-B51 121 (SEQ. ID NO.:121) EBV EBNA-6 213-222 QNGALAINTFHLA-1362 112 (SEQ. ID NO.:122) EBV EBNA-3 603-611 RLRAEAGVK HLA-A3 123(SEQ. ID NO.:123) HIV sAg 348-357 GLSPTVWLSV HLA-A*0201 124 (SEQ. IDNO.:124) HBV SAg 335-343 WLSLLVPFV HLA-A*0201 124 (SEQ. ID NO.:125) HBVcAg 18-27 FLPSDFFPSV HLA-A*0201125, 126, 127 (SEQ. ID NO.:126) REV cAg18-27 FLPSDFFPSV HLA-A*0202 127 (SEQ.ID NO.:126) REV cAg 18-27FLPSDFFPSV HLA-A*0205 127 (SEQ. ID NO.:126) REV cAg 18-27 FLPSDFFPSVHLA-A*0206 127 (SEQ. ID NO.:126) HCV MP 105-112 ILHTPGCV HLA-A*0201 139(SEQ. ID NO.:151) HCV env E 66-75 QLRRHIDLLV HLA-A*0201 139 (SEQ. 1DNO.:152) HCV env E 88-96 DLCGSVFLV HLA-A*0201 139 (SEQ. TD NO.:153) HCVenv E 172-180 SMVGNWAKV HLA-A*0201 139 (SEQ. ID NO.:154) HCV NS1 308-316HLIIQNIVDV HLA-A*0201 139 (SEQ. ID NO.:155) HCV NS1 340-348 FLLLADARVHLA-A*0201 139 (SEQ. ID NO.:156) HCV NS2 234-246 GLRDLAVAVEPVVHLA-A*0201 139 (SEQ. ID NO.:157) HCV NS1 18-28 SLLAPGAKQNV HLA-A*0201139 (SEQ. ID NO.:158) HCV NS1 19-28 LLAPGAKQNV HLA-A*0201 139 (SEQ. IDNO.:159) HCV NS4 192-201 LLFNILGGWV HLA-A*0201 129 (SEQ. ID NO.:160) HCVNS3 579-587 YLVAYQATV HLA-A*0201 129 (SEQ. ID NO.:161) HCV core protein34-43 YLLPRRGPRL HLA-A*0201 129 (SEQ. ID NO.:162) HCV MP 63-72LLALLSCLTI HLA-A*0201 129 (SEQ. ID NO.:163) HCV NS4 174-182 SLMAFTAAVHLA-A*0201 140 (SEQ. ID NO.:164) HCV NS3 67-75 C1NGVCWTV HLA-A*0201 140(SEQ. ID NO.:165) HGV NS3 163-171 LLGPAGHAV HLA-A*0201 141 (SEQ.IDNO.:166) HCV NS5 239-247 ILDSFDPLV HLA-A*0201 141 (SEQ. ID NO.:167) HCVNS4 A236-244 ILAGYGAGV HLA-A*0201 142 (SEQ. ID NO.:168) HCV NS5 714-722GLQDCTMLV HLA-A*0201 142 (SEQ. ID NO.:169) HCV NS3 281-290 TGAPVTYSTYHLA-A*0201 143 (SEQ. ID NO.:170) HCV NS4 A149-157 HMWNFISGI HLA-A*0201144 (SEQ. ID NO.:171) HCV NS5 575-583 RVCEKMALY HLA-A*0201 145 A3 (SEQ.ID NO.:172) HCV NS1 238-246 TINYTIFK HLA-A*1101 145 (SEQ. ID NO.:173)HCV NS2 109-116 YISWCLWW HLA-A23 145 (SEQ. ID NO.:174) HCV core protein40-48 GPRLGVRAT HLA-B7 145 (SEQ.ID NO.:175) HIV-1 gp120 380-388SFNCGGEFF HLA-Cw*0401 185 (SEQ.ID NO.:176) HIV-1 RT 206-214 TEMEKEGKIH2-Kk 186 HIV-1 p17 18-26 KIRLRPGGK HLA-A*0301 187 (SEQ. ID NO.:178)HIV-1 P17 20-29 RLRPGGKKKY HLA-A*030l 188 (SEQ. ID NO.:179) HIV-1 RT325-333 AIFQSSMTK HLA-A*0301 188 (SEQ..LD NO.:180) HIV-1 p17 84-92TLYCVHQRI HLA-A11 188 (SEQ. ID NO.:181) HIV-1 RT 508-517 IYQEPFKNLKHLA-A11 188 (SEQ. ID NO.:182) HIV-1 p17 28-36 KYKLKIIIVW HLA-A24 188(SEQ. ID NO.:183) HIV-1 gp120 53-62 LFCASDAKAY HLA-A24 189 (SEQ. IDNO.:184) HIV-1 gagp24 145-155 QAISPRTLNAW HLA-A25 188 (SEQ. ID NO.:185)HIV-1 gagp24 167-175 EVIPMFSAL HLA-A26 188 (SEQ. ID NO.:186) HIV-1 RT593-603 ETFYVDGAANR HLA-A26 188 (SEQ. ID NO.:187) HIV-1 gp41 775-785RLRDLLLIVTR HLA-A31 190 (SEQ.ID NO.:188) HIV-1 RT 559-568 PIQKETWETWHLA-A32 187 (SEQ. ID NO.:189) HIV-1 gp120 419-427 RIKQIINMW HLA-A32 187(SEQ.ID NO.:190) HIV-1 RT 71-79 ITLWQRPLV HLA-A*6802 188 (SEQ.IDNO.:191) HIV-1 RT 85-93 DTVLEEMNL HLA-A*6802 188 (SEQ. ID NO.:192) HIV-1RT 71-79 ITLWQRPLV HLA-A*7401 188 (SEQ. ID NO.:193) HIV-1 gag p24148-156 SPRTLNAWV HLA-B7 188 (SEQ. ID NO.:194) HIV-1 gagp24 179-187ATPQDLNTM HLA-B7 188 (SEQ. ID NO.:195) HIV-1 gp120 303-312 RPNNNTRKSIHLA-B7 188 (SEQ. ID NO.:196) HIV-1 gp41 843-851 IPRRIRQGL HLA-B7 188(SEQ.ID NO.:197) HIV-1 p17 74-82 ELRSLYNTV HLA-B8 188 (SEQ. ID NO.:198)HIV-1 nef 13-20 WPTVRERM HLA-B8 188 (SEQ. ID NO.:199) HIV-1 nef 90-97FLKEKGGL HLA-B8 188 (SEQ. ID NO.:200) HIV-1 gag p24 183-191 DLNTMLNTVHLA-B14 191 (SEQ. ID NO.:568) HIV-1 P17 18-27 KIRLRPGGKK HLA-B27 188(SEQ. ID NO.:201) HIV-1 p17 19-27 IRLRPGGKK HLA-B27 188 (SEQ. IDNO.:202) HIV-1 gp41 791-799 GRRGWEALKY HLA-B27 188 (SEQ. ID NO.:203)HIV-1 nef 73-82 QVPLRPMTYK HLA-B27 188 (SEQ. ID NO.:204) HIV-1 GP41590-597 RYLKDQQL HLA-B27 192 (SEQ. ID NO.:205) HIV-l nef 105-114RRQDILDLWI HLA-B*2705 188 (SEQ. ID NO.:206) HIV-1 nef 134-141 RYPLTFGWHLA-B*2705 188 (SEQ. ID NO.:207) HIV-1 p17 36-44 WASRELERF HLA-B35 188(SEQ. ID NO.:208) HIV-1 GAG P24 262-270 TVLDVGDAY HLA-B35 188 (SEQ. IDNO.:209) HIV-1 gp120 42-52 VPVWKEAT1T~L HLA-B35 188 (SEQ. ID NO.:210)HIV-1 P17 36-44 NSSKVSQNY HLA-B35 193 (SEQ. ID NO.:221) HIV-1 gag p24254-262 PPIPVGDIY HLA-B35 193 (SEQ. ID NO.:212) HIV-1 RT 342-350HPDIVIYQY HLA-B35 193 (SEQ. ID NO.:213) HIV-1 gp41 611-619 TAVPWNASWHLA-B35 194 (SEQ. ID NO.:214) HIV-1 gag 245-253 NPVPVGN1Y HLA-B35 193(SEQ. ID NO.:215) HIV-1 nef 120-128 YFPDWQNYT HLA-B37 188 (SEQ. IDNO.:216) HIV-1 gag p24 193-201 GHQAAMQML HLA-B42 188 (SEQ. ID NO.:217)HIV-1 p17 20-29 RLRPGGKXIKY HLA-B42 188 (SEQ. ID NO.:218) Thy-1 RT438-446 YPGIKVRQL HLA-B42 188 (SEQ. ID NO.:219) HIV-1 RT 591-600GAETFYVDGA HLA-B45 188 (SEQ. ID NO.:220) HIV-1 gag p24 325-333 NANPDCKTIHLA-BS1 188 (SEQ. ID NO.:221) HIV-1 gag p24 275-282 RMYSPTSI HLA-B52 188(SEQ. ID NO.:222) HIV-1 gp120 42-51 VPVWKEATIT HLA-B*5501 192 (SEQ. IDNO.:223) HIV-1 gag p24 147-155 ISPRTLNAW HLA-B57 188 (SEQ. ID NO.:224)HIV-1 gag p24 240-249 TSTLQEQIGW HLA-B57 188 (SEQ. ID NO.:225) HIV-1 gagp24 162-172 KAFSPEVIPMF HLA-B57 188 (SEQ. ID NO.:226) HIV-1 gagp24311-319 QASQEVKNW HLA-B57 188 (SEQ. ID NO.:227) H1V-1 gagp24 311-319QASQDVKNW HLA-B57 188 (SEQ. ID NO.:228) HIV-1 nef 116-125 HTQGYEPDWQHLA-B57 188 (SEQ. ID NO.:229) HIV-1 nef 120-128 YFPDWQNYT HLA-B57 188(SEQ. ID NO.:230) HIV-1 gag p24 240-249 TSTLQEQIGW HLA-B58 188 (SEQ. IDNO.:231) HIV-1 p17 20-29 RLRPGGKKKY HLA-B62 188 (SEQ. ID NO.:232) HIV-1p24 268-277 LGLNKJVRMY HLA-B62 188 (SEQ. ID NO.:233) HIV-1 RT 415-426LVGKLNWASQIY HLA-B62 188 (SEQ. ID NO.:234) HIV-1 RT 476-485 ILKEPVHGVYHLA-B62 188 (SEQ. ID NO.:235) HIV-1 nef 117-127 TQGYFPDWQNY HLA-B62 188(SEQ. ID NO.:236) HIV-1 nef 84-91 AVDLSIffL HLA-B62 188 (SEQ. IDNO.:237) HIV-1 gag p24 168-175 VIPMFSAL HLA-Cw*0102 188 (SEQ. IDNO.:238) HIV-1 gp120 376-384 FNCGGEFFY HLA-A29 196 (SEQ. ID NO.:239)HIV-1 gp120 375-383 SFNCGGEFF HLA-B15 196 (SEQ. ID NO.:240) HIV-1 nef136-145 PLTFGWCYKL HLA-A*0201 197 (SEQ. ID NO.:241) HIV-1 nef 180-189VLEWRFDSRL HLA-A*0201 197 (SEQ. ID NO.:242) HIV-1 nef 68-77 FPVTPQVPLRHLA-B7 197 (SEQ. ID NO.:243) HIV-1 nef 128-137 TPGPGVRYPL HLA-B7 197(SEQ. ID NO.:244) HIV-1 gag p24 308-316 QASQEVKNW HLA-Cw*0401 521 (SEQ.ID NO.:245) HIV-1 IIIB RT 273-282 TPLDEDFRKY HLA-B35 181 (SEQ. IDNO.:246) HIV-1 IIIB RT 25-33 NPDIVIYQY HLA-B35 181 (SEQ. ID NO.:247)HIV-1 IIIB gp41 557-565 RAIEAQALIL HLA-B51 181 (SEQ. ID NO.:248) HIV-1IIIB RT 231-238 TAFTIPSI HLA-B51 181 (SEQ. ID NO.:249) HIV-1 IIIB p24215-223 VHPVHAGPIA HLA-B*5501 181 (SEQ. ID NO.:250) HIV-1 IIIB gp120156-165 NCSFNISTSI HLA-Cw8 181 (SEQ. ID NO.:251) HIV-1 IIIB gp120241-249 CTNVSTVQC HLA-Cw8 181 (SEQ. ID NO.:252) HIV-1 5F2 gp120 312-320IGPGRAFHT H2-Dd 198 (SEQ. ID NO.:253) HIV-1 5F2 pol 25-33 NPDWIYQYHLA-B*3501 199 (SEQ. ID NO.:254) HIV-1 5F2 pol 1432-441 EPIVGAETFYHLA-B*3501 199 (SEQ. ID NO.:255) HIV-1 5F2 pol 432-440 EPWGAETFHLA-B*3501 199 (SEQ. ID NO.:256) HIV-1 5F2 pol 6-14 SPAIFQSSM HLA-B*3501199 (SEQ. ID NO.:257) HIV-1 5F2 pol 59-68 VPLDKDFRKY HLA-B*3501 199(SEQ. ID NO.:258) HIV-1 5F2 pol 6-14 IPLTEEAEL HLA-B*3501 199 (SEQ. IDNO.:259) HIV-1 5F2 nef 69-79 RPQVPLRPMTY HLA-B*3501 199 (SEQ. IDNO.:260) HIV-1 5F2 nef 66-74 FPVRPQVPL HLA-B*3501 199 (SEQ. ID NO.:261)HIV-1 5F2 env 10-18 DPNPQEVVL HLA-B*3501 199 (SEQ. ID NO.:262) HIV-1 5F2env 7-15 RPIVSTQLL HLA-B*3501 199 (SEQ. ID NO.:263) HIV-1 5F2 p01 6-14IPLTEEAEL HLA-BS1 199 (SEQ. ID NO.:264) HIV-1 5F2 env 10-18 DPNPQEVVLHLA-B51 199 (SEQ. ID NO.:265) HIV-1 5F2 gagp24 199-207 AMQMLKETI H2-Kd198 (SEQ. ID NO.:266) HIV-2 gagp24 182-190 TPYDrNQML HLA-B*5301 200(SEQ. ID NO.:267) HIV-2 gag 260-269 RRWIQLGLQKV HLA-B*2703 188 (SEQ. IDNO.:268) HIV-1 5F2 gp41 593-607 GIWGCSGKLICTFA HLA-B17 201 V (SEQ. IDNO.:269) HIV-1 5F2 gp41 753-767 ALIWEDLRSLCLFSY HLA-B22 201 (SEQ. IDNO.:270) HPV 6b E7 21-30 GLHCYEQLV HLA-A*0201 202 (SEQ. ID NO.:271) HPV6b E7 47-55 PLKQHFQW HLA-A*0201 202 (SEQ. ID NO.:272) HPV11 E7 4-12RLVTLKDIV HLA-A*0201 202 (SEQ. ID NO.:273) HPV16 E7 86-94 TLGWCPICHLA-A*0201 129 (SEQ. ID NO.:274) HPV16 E7 85-93 GTLGWCPI HLA-A*0201 129(SEQ. ID NO.:275) HPV16 E7 12-20 MLDLQPETI HLA-A*0201 129 (SEQ. IDNO.:276) HPV16 E7 11-20 YMLDLQPETT HLA-A*0201 203 (SEQ. ID NO.:277)HPV16 E6 15-22 RPRIKLPQL HLA-B7 204 (SEQ. ID NO.:278) HPV16 E6 49-57RAHYNIVTF HW-Db 205 (SEQ. ID NO.:279) HSV gp B 498-505 SSIEFARL 112-Kb206 (SEQ. ID NO.:280) HSV-1 gp C 480-488 GIGIGVLAA HLA-A*0201 104 (SEQ.ID NO.:281) HSV-1 ICP27 448-456 DYATLGVGV H2-Kd 207 (SEQ. ID NO.:282)HSV-1 ICP27 322-332 LYRTFAGNPRA H2-Kd 207 (SEQ. ID NO.:283) HSV-1 UL39822-829 QTFDFGRL H2-Kb 208 (SEQ. ID NO.:284) HSV-2 gpC 446-454 GAGIGVAVLHLA-A*0201 104 (SEQ. ID NO.:285) HLTV-1 TAX 11-19 LLFGYPVYV HLA-A*0201209 (SEQ. ID NO.:286) Influenza NIP 58-66 GILGFVFTL HLA-A*020 168, 169,209, 210, 211 (SEQ. ID NO.:287) Influenza MP 59-68 ILGFVFTLTVHLA-A*0201168, 212, 213 (SEQ. ID NO.:288) Influenza NP 265-273 ILRGSVAHKHLA-A3 214 (SEQ. ID NO.:289) Influenza NP 91-99 KTGGPIYKR HLA-A*6801215, 216 (SEQ. ID NO.:290) Influenza NP 380-388 ELRSRYWAI HLA-B8 217(SEQ. ID NO.:291) Influenza NP 381-388 LRSRYWAI HLA-B*2702 218 (SEQ. IDNO.:292) Influenza NP 339-347 EDLRVL5FI HLA-B*3701 219 (SEQ. ID NO.:293)Influenza NSI 158-166 GEISPLPSL HLA-B44 220 (SEQ. ID NO.:294) InfluenzaNP 338-346 FEDLRVL5F HLA-B44 220 (SEQ. ID NO.:295) Influenza NSI 158-166GEISPLPSL HLA-B*44O2 220 (SEQ. ID NO.:294) Influenza NP 338-346FEDLRVL5F HLA-B*4402 220 (SEQ. ID NO.:295) Influenza PBI 591-599VSDGGPKLY lILA-Al 214, 29 (SEQ. ID NO.:296) Influenza A NP 44-52CTELKLSDY HLA-Al 29 (SEQ. ID NO.:297) Influenza NSI 122-130 AIMDKNIILHLA-A*0201 221 (SEQ. ID NO.:298) Influenza A NSI 123-132 IMDKNIILKAHLA-A*0201 221 (SEQ. ID NO.:299) Influenza A NP 383-391 SRYWAIRTRHLA-B*2705 160, 184 (SEQ. ID NO.:300) Influenza A NP 147-155 TYQRTRALVH2-Kd 222, 223 (SEQ. ID NO.:301) Influenza A HA 210-219TYVSVSTSTL H2-Kd224, 225 (SEQ. ID NO.302) Influenza A HA 518-526 IYSTVASSL H2-Kd 224(SEQ. ID NO.303) Influenza A HA 259-266 FEANGNLI H2-Kk 226, 227, 228(SEQ. ID NO.:304) Influenza A HA 10-18 IEGGWTGM1 H2-Kk 226, 227, 228(SEQ. ID NO.:305) Influenza A NP 50-57 SDYEGRLI H2-Kk 229, 230 (SEQ. IDNO. 306) Influenza a NSI 152-160 EEGAIVGEI H2-Kk 231 (SEQ. ID NO.:307)Influenza A34 NP 36-374 ASNENMETM H2Db 168, 222, 219 (SEQ. ID NO.:308)Influenza A68 NP 366-374 ASNENMDAM H2Db 232 (SEQ. ID NO.:3 09) InfluenzaB NP 85-94 KLGEFYNQMM HLA-A*0201 233 (SEQ. ID NO.:310) Influenza B NP85-94 KAGEFYNQMM HLA-A*0201 234 (SEQ. ID NO.:311) Influenza JAP HA204-212 LYQNVGTYV H2Kd 235 (SEQ. ID NO.312) Influenza JAP HA 210-219TYVSVGTSTL H2-Kd 225 (SEQ. ID NO.:313) Influenza JAP HA 523-531VYQILATYA H2-Kd 235 (SEQ. ID NO.314) Influenza JAP HA 529-537 IYATVAGSLH2-Kd 235 (SEQ. ID NO.315) Influenza JAP HA 210-219 TYVSVGTSTI(L > I)H2-Kd 236 (SEQ. ID NO.:316) Influenza TAP HA 255-262 FESTGNLI H2-Kk 237(SEQ. ID NO.:317) JHMV cAg 318-326 APTAGAFFF H2-Ld 238 (SEQ. ID NO.:318)LCMV NP 118-126 RPQASGVYM H2-Ld 239-240 (SEQ. ID NO.319) LCMV NP396--404 FQPQNGQFI H2-Db 241 (SEQ. ID NO.320) LCMV GP 276-286SGVENPGGYCL H2-Db 242 (SEQ. ID NO.:321) LCMV GP 33-42 KAVYNFATCG H2-Db243, 244 (SEQ. ID NO.:322) MCMV pp89 168-176 YPHFMPTNL H2-Ld 245 (SEQ.ID NO.323) MHV spike protein 510-518 CLSWNGPHL H2-Db 248 (SEQ. IDNO.324) MMTV env gp13 6474-482 5FAVATTAL H2-Kd 246 (SEQ. ID NO.:325)MMTV gag p27 425-433 SYETFISRL H2-Kd 246 (SEQ. ID NO.:326) MMTV env gp73544-551 ANYDFICV H2-Kb 247 (SEQ. ID NO.:327) MuLV env piSE 574-58 1KSPWFTTL H2-Kb 249, 250 (SEQ. ID NO.:328) MuLV env gp70 189-196 SSWDFITV112-Kb251, Sijts et al. Submitted (SEQ. ID NO.:329) MULV gag 75K 75-83CCLCLTVFL H2-Db 252 (SEQ. ID NO.:330) MuLV env gp70 423-431 SPSYVYHQFH2Ld 253 (SEQ. ID NO.:331) MV F protein 437-447 SRRYPDAVYLH HLA~B*2705254 (SEQ. ID NO.:332) Mv F protein 438-446 RRYPDAVYL HLA-B*2705 255(SEQ. ID NO.333) Mv NP 281-289 YPALGLHEF H2-Ld 256 (SEQ. ID NO.:334) MvHA 343-351 DPVIDRLYL H2-Ld 257 (SEQ. ID NO.335) MV HA 544-552 SPGR5FSYFH2-Ld 257 (SEQ. ID NO.:336) Poliovirus VP1 111-118 TYKDTVQL H2-kd 258(SEQ. ID NO.:337) Poliovirus VP1 208-217 FYDGFSKVPL H2-Kd 258 (SEQ. IDNO.:338) Pseudorabies G111 455-463 IAGIGILAI HLA-A*O201 104 virus gp(SEQ. ID NO.:339) Rabiesvirus NS 197-205 VEAEBNAHQI H2-Kk 227-227 (SEQ.ID NO.:340) Rotavirus VP7 33-40 IIYRELL1 H2-Kb 259 (SEQ. ID NO.:341)Rotavirus VP6 376-384 VGPVFPPGM 112-Kb 260 (SEQ. ID NO.:342) RotavirusVP3 585-593 YSGYIFRDL 112-Kb 260 (SEQ. ID NO.:343) RSV M2 82-90SYIGSINNI H2-Kd 261 (SEQ. ID NO.:344) SIV gagp11C 179-190 EGCTPYDTNQMLMamu-A*01 266 (SEQ. ID NO.:345) SV NP 324-332 FAPGNYPAL H2-Db 262 (SEQ.ID NO.:346) SV NP 324-332 FAPCTNYPAL 112-Kb263, 264, 265 (SEQ. IDNO.:346) 5V40 T 404-411 VVYDFLKC 112-Kb 267 (SEQ. ID NO.:347) 5V40 T206-215 SAINNYAQKL H2-Db 268, 269 (SEQ. ID NO.:348) 5V40 T 223-231CKGVNKEYL H2-Db 268, 269 (SEQ. ID NO.349) 5V40 T 489-497 QGINNLDNL H2-Db268, 269 (SEQ. ID NO.:350) 5V40 T 492-500 NNLDNLRDY(L) H2-Db 270 (501)(SEQ. ID NO.:351) 5V40 T 560-568 SEFLLEKRI H2-Kk 271 (SEQ. ID NO.352)VSV NP 52-59 RGYVYQGL 112-Kb 272 (SEQ. ID NO.:353)

[0101] Table II sets forth antigens identified from various proteinsources. The Table is extracted from Table 4.2 in the Rammensee bookwith the references in Table H being the same as the references in theRammensee Table 4.2. TABLE II HLA Class I Motifs HLA-A1 Position(Antigen) Source Ref. T cell epitopes EADPTGHSY MAGE-1 161-169 27, 28(SEQ. ID NO.:354) VSDGGPNLY Influenza A PB 1591-599 21, 23 (SEQ. IDNO.:355) CTELKLSDY Influenza A NP 44-52 23 (SEQ. ID NO.:356) EVDPIGHLYMAGE-3 168-176 29, 30 (SEQ. ID NO.:357) HLA-A201 MLLSVPLLLG Calreticulinsignal sequence I-10 34, 35, 36, 37 (SEQ. ID NO.:358) STBXQSGXQ HBVPRE-S PROTEIN 141-149 43 (SEQ. ID NO.:359) YMDGTMSQV Tyrosinase 369-37745 (SEQ. ID NO.:360) ILKEPVHGV HIV-I RT 476-484 4, 31, 47 (SEQ. IDNO.:361) LLGFVFTLTV Influenza MP 59-68 4, 39 (SEQ. ID NO.:362)LLFGYPVYVV HTLV-1 tax 11-19 40 (SEQ. ID NO.:363) GLSPTVWLSV HBV sAg348-357 48 (SEQ. ID NO.:364) WLSLLVPFV HBV sAg 335-343 49, 50, 51 (SEQ.ID NO.:365) (SEQ. ID NO.:366) CLG0LLTMV EBVLMP-2 426-434 48 (SEQ. IDNO.:367) FLAGNSAYEYV HCMV gp 618-628B 53 (SEQ. ID NO.:368) KLGEFYNQMMInfluenza BNP 85-94 54 (SEQ. ID NO.:369) KLVALGINAV HCV-1 N53 400-409 55(SEQ. ID NO, :370) DLMGYIPLV HCV MP 17-25 56 (SEQ. ID NO.:371) RLVTLKDIVHPV 11 EZ 4-12 34, 35 (SEQ. ID NO.:372) MLLAVLYCL Tyrosinase 1-9 57, 58,59, 68 (SEQ. ID NO-373) AAGJGILTV Melan A/Mart-127-35 60 (SEQ. IDNO.:374) YLEPGPVTA Pmel 17/gp 100 480-488 61 (SEQ. ID NO.:375)ILDGTATLRL Pmel 17/gp 100 457-466 62 (SEQ. ID NO.:376) LLDGTATLRL Pmelgp1OO 457-466 62 (SEQ. lID NO.:377) ITDQVPFSV Pmel gp 100 209-217 62(SEQ. ID NO.:378) KTWGQYWQV Pmel gp 100 154-162 62 (SEQ. ID NO.:379)TITDQVPFSV Pmel gp 100 208-217 62 (SEQ. ID NO.:380) AFHILVAREL HIV-Inef190-198 63 (SEQ. ID NO.:381) YLNKJQNSL P. falciparum CSP 334-342 64(SEQ. ID NO.:382) MMRKLAILSV P. falciparum CSP 1-10 64 (SEQ. ID NO.:383)KAGEFYNQMM Influenza BNP 85-94 65 (SEQ. ID NO.:384) NIAEGLRAL EBNA-1480-488 66 (SEQ. ID NO.:385) NLRRGTALA EBNA-1 519-527 66 (SEQ. IDNO.:386) ALAIPQCRL EBNA-1 525-533 66 (SEQ. ID NO.:387) VLKDATKDL EBNA-1575-582 66 (SEQ. ID NO.:388) FMVFLQTHI EBNA-1 562-570 66 (SEQ. IDNO.:389) HLIVDTDSL EBNA-2 15-23 66 (SEQ. ID NO.:390) SLGNPSLSV EBNA-222-30 66 (SEQ. ID NO.391) PLASAMRML EBNA-2 126-134 66 (SEQ. ID NO.392)RMLWMANYI EBNA-2 132-140 66 (SEQ. ID NO.:393) MLWMANYIV EBNA-2133-141 66(SEQ. ID NO.:394) RPQGPQTA EBNA-2 151-159 66 (SEQ. ID NO.:395)PLRPTAPTTI EBNA-2 171-179 66 (SEQ. ID NO.:396) PLPPAThTV EBNA-2 205-21366 (SEQ. ID NO.:397) RMHLPVLHV EBNA-2 246-254 66 (SEQ. ID NO.397)PMPLPPSQL EBNA-2 287-295 66 (SEQ. ID NO.:399) QLPPPAAPA EBNA-2 294-30266 (SEQ. ID NO.:400) SMPELSPVL EBNA-2 381-389 66 (SEQ. ID NO.:401)DLDESWDY1 EBNA-2 453-461 66 (SEQ. ID NO.:402) PLPGVLWPVV BZLF1 43-51 66(SEQ. ID NO.:403) SLEECDSEL BZLF1 167-175 66 (SEQ. ID NO.:404) EIKRYKNRVBZLFI 176-184 66 (SEQ. ID NO.:405) QLLQFIYREV BZLF1 195-203 66 (SEQ. IDNO.:406) LLQHYREVA BZLFI 196-204 66 (SEQ. ID NO.:407) LLKQMCPSL BZLFI217-225 66 (SEQ. ID NO.:408) SIIPRTPDV BZLFI 229-237 66 (SEQ. IDNO.:409) AIMDKNIIL Influenza A NS1 122-130 67 (SEQ. ID NO.:410)IMDKNLILKA Influenza A NS1 123-132 67 (SEQ. ID NO.411) LLALLSCLTV HCV MP63-72 69 (SEQ. ID NO.:412) ILHTPGCV HCVMP 105-112 69 (SEQ. ID NO.:413)QLRRIIIDLLV HCV env E 66-75 69 (SEQ. ID NO.:414) DLCGSVFLV HCV env E88-96 69 (SEQ. ID NO.:415) SMVGNWAKV HCV env E 172-180 69 (SEQ. IDNO.:416) HLHQNIVDV HCV NSI 308-316 69 (SEQ. ID NO.:417) FLLLADARV HCVNSI 340-348 69 (SEQ. ID NO.:418) GLRDLAVAVEPVV HCV NS2 234-246 69 (SEQ.ID NO.:419) SLLAPGAKQNV HCV NS1 18-28 69 (SEQ. ID NO.:420) LLAPGAiKQNVHCV NS1 19-28 69 (SEQ. ID NO.:421) FLLSLGIIIL HBV pol 575-583 70 (SEQ.ID NO.:422) SLYADSPSV HBV pol 816-824 70 (SEQ. ID NO.:423) GLSRYVARLHIBV POL 455-463 70 (SEQ. ID NO.:424) KIFGSLAFL HER-2 369-377 71 (SEQ.ID NO.:425) ELVSEFSRM HER-2 971-979 71 (SEQ. ID NO.:426) KLTPLCVTL HIV-Igp 160 120-128 72 (SEQ. ID NO.:427) SLLNATDIAV HIV-I GP 160 814-823 72(SEQ. ID NO.:428) VLYRYG5FSV Pmel gp100 476-485 62 (SEQ. ID NO.:429)YIGEVLVSV Non-filament forming class I 73 myosin family (HA-2)** (SEQ.ID NO.:430) LLFNILGGWV HCV NS4 192-201 74 (SEQ. ID NO.:431) LLVPFVQWFWHBV env 338-347 74 (SEQ. ID NO.:432) ALMPLYACI HBV pol 642-650 74 (SEQ.ID NO.:433) YLVAYQATV HCV NS3 579-587 74 (SEQ. ID NO.:434) TLGIVCPICHIPV 16 E7 86-94 74 (SEQ. ID NO.:435) YLLPRRGPRL HCV core protein 34-4374 (SEQ. ID NO.:436) LLPIFFGLWV HBV env 378-387 74 (SEQ. ID NO.:437)YMDDVVLGA HBV Pol 53 8-546 74 (SEQ. ID NO.:438) GTLGIVCPI HPV16 E7 85-9374 (SEQ. ID NO.:439) LLALLSCLTI HCY MP 63-72 74 (SEQ. ID NO.:440)MLDLQPETf HPV 16 E7 12-20 74 (SEQ. ID NO.:441) SLMAFTAAV HCV N54 174-18275 (SEQ. ID NO.:442) C1NGVCWTV HCV NS3 67-75 75 (SEQ. ID NO.:443)VIVINILLQYVV Glutamic acid decarboxylase 76 114-123 (SEQ. ID NO.:444)ILTVILGVL Melan A/Mart- 32-40 77 (SEQ. ID NO.:445) FLWGPRALV MAGE-3271-279 78 (SEQ. ID NO.:446) LLCPAGHAV HCV NS3 163-171 54 (SEQ. IDNO.:447) ILD5FDPLV HCV NSS 239-247 54 (SEQ. ID NO.:448) LLLCLIFLL HBVenv 250-258 79 (SEQ. ID NO.:449) LIDYQGMLPV HBV env 260-269 79 (SEQ. IDNO.:450) SIVSPFIPLL HBV env 370-379 79 (SEQ.ID NO.:451) FLLTRILTI HBVenv 183-191 80 (SEQ. ID NO.:452) HLGNVKYLV P. faciparum TRAP 3-11 81(SEQ. ID NO.:453) GIAGGLALL P. faciparum TRAP 500-508 81 (SEQ. IDNO.:454) ILAGYGAGV HCV NS 54A 23 82 (SEQ. ID NO.:455) 6-244 GLQDCTMLVHCV NS5 714-722 82 (SEQ. ID NO.:456) TGAPVTYSTY HCV NS3 281-290 83 (SEQ.ID NO.:457) VIYQYMDDLV HIV-1RT 179-187 84 (SEQ. ID NO.:458) VLPDVFIRCVN-acetylglucosaminyl- 85 transferase (SEQ. ID NO.:459) V Gnt-V intronVLPDVFIRC N-acetylglucosaminyl- 85 transferase (SEQ. ID NO.:460) V Gnt-Vintron AVGIGIAVV Human CD9 86 (SEQ. ID NO.:461) LVVLGLLAV Humanglutamyl- 86 (SEQ. ID NO.:462) transferase ALGLGLLPV Human G protein 86coupled (SEQ. ID NO.:463) receptor 164-172 GIGIGVLAA HSV-I gp C 480-48886 (SEQ. ID NO.:281) GAGIGVAVL HSV-2 gp C 446-454 86 (SEQ. ID NO.:464)JAGIGILAI Pseudorabies gpGIN 86 (SEQ. ID NO.:465) 455-463 LIVIGILILAdenovirus 3 E3 9kD 86 (SEQ. ID NO.:466) 30-38 LAGIGLIAA S. LincolnensisImrA 86 (SEQ. ID NO.:467) VDGIGILTI Yeast ysa-1 77-85 86 (SEQ. IDNO.:468) GAGIGVLTA B. polymyxa, 86 149- βcndoxylanase (SEQ. ID NO.:469)157 AAGIGIIQI E. coli methionine 86 590-598 synthase (SEQ. ID NO.:470)QAGIGILLA E. coli hypothetical 86 (SEQ. ID NO.:471) protein 4-12KARDPHSGHFV CDK4wl 22.32 87 (SEQ. ID NO.:472) KACDPI-ISGIIFV CDK4-R24C22-32 87 (SEQ. ID NO.:473) ACDPFISGHFV GDK4-R24C 23-32 87 (SEQ. IDNO.:474) SLYNTVATL HIV- I gag p 17 77-85 99 (SEQ. ID NO.:475) ELVSEFSRVHER-2, m > V 89 971-979 substituted (SEQ. ID NO.:476) RGPGRAFVTI HIV-Igp 160 315-329 90 (SEQ. ID NO.:477) HMWNFISGI HCV NS4A 149-157 91 (SEQ.ID NO.:478) NLVPMVATVQ HCMV pp65 495-504 92 (SEQ. ID NO.:479) GLHCYEQLVHPV 6b E7 21-30 93 (SEQ. ID NO, :480) PLKQIJFQIV HPV 6b E7 93 (SEQ. IDNO.:481) 47-55 LLDFVRfMGV EBNA-6 284-293 95 (SEQ. ID NO.:482) ATMEKNIMLInfluenza Alaska NS 1 122-130 67 (SEQ. ID NO.:483) YLKTIQNSL P.falciparum cp36 CSP 96 (SEQ. ID NO.:484) YLNKIQNSL P. falciparum cp39CSP 96 (SEQ. ID NO.:485) YMLDLQPE1T HPV 16 E7 11-20* 97 (SEQ. IDNO.:486) LLMGTLGIV HPV16 E7 82-90** 97 (SEQ. ID NO.:487) TLGIVCPI HPV 16E7 86-93 97 (SEQ. ID NO.:488) TLTSCNTSV HIV-1 gp120 197-205 98 (SEQ. IDNO.:489) KLPQLCTEL HPV 16 E6 18-26 97 (SEQ. ID NO.:490) THIDITLEG HPV16E6 29-37 97 (SEQ. ID NO.:491) LGIVCPICS HPV16 E7 87-95 97 (SEQ. IDNO.:492) VILGVLLLI Melan A/Mart-1 35-43 68 (SEQ. ID NO.:493) ALMDKSLHVMelan A/Mart-1 56-64 68 (SEQ. ID NO.:494) GILTVILGV Melan A/Mart-1 31-3968 (SEQ. ID NO.:495) T cell epitopes M1NAYLDKL P. Falciparum STARP 81(SEQ. ID NO.:496) 523-531 AAGIGILTV Melan A/Mart- 127-35 100 (SEQ. IDNO.:497) FLPSDFFPSV HBV cAg 18-27 51 (SEQ. ID NO, :498) Motif unknownSVRDRLARL EBNA-3 464-472 101 T cell epitopes (SEQ. ID NO.:499) T cellepitopes AAGIGILTV Melan A/Mart-1 27-35 100 (SEQ. ID NO.:497) FAYDGKDYIHuman MHC I-ot 99 (SEQ. ID NO.:500) 140-148 T cell epitopes AAGIGILTVMelan A/Mart-1 27-35 100 (SEQ. ID NO.:497) FLPSDFFPSV HBV cAg 18-27 51(SEQ. ID NO.:498) Motif unknown AAGIGILTV Meland A/Mart-1 100 T cellepitopes (SEQ. ID NO.:497) 27-35 FLPSDFFPSV HBV cAg 18-27 51 (SEQ. IDNO.:498) AAGIGILTV Melan A/Mart-1 27-35 100 (SEQ. ID NO.:497) ALLAVGATKPmell7 gp 100 17-25 107 (SEQ. ID NO.:501) T cell epitopes RLRDLLLIVTRHIV-1 gp41 768-778 108 (SEQ. ID NO.:502) QVPLRPMTYK HIV-1 nef 73-82 109(SEQ. ID NO.:503) TVYYGVPVWK HIV-1 gp120-36-45 110 (SEQ. ID NO.:504)RLRPGGKKK HIV-1 gagp 17 20-29 110 (SEQ. ID NO.:505) ILRGSVAHK InfluenzaNP 265-273 21 (SEQ. ID NO, :506) RLRAEAGVK EBNA-3 603-611 111 (SEQ. IDNO.:507) RLRDLLLIVTR HIV-1 gp41 770-780 112 (SEQ. ID NO.:502) VYYGVPVWKHIV-I GP 120 38-46 113 (SEQ. ID NO.:508) RVCEKMALY HCV N55 575-583 114(SEQ. ID NO.:509) Motif unknown KIFSEVTLK Unknown; muta melanoma Wolfelpeptide ted (p I 83L) et al., T cell epitope (SEQ. ID NO.:510) 175-183pers. Comm. YVNVNMGLK* HBV cAg 88-96 116 (SEQ. ID NO.:511) T cellepitopes IVTDFSVIK EBNA-4 416-424 115, (SEQ. ID NO.:512) 117 ELNEALELKP53 343-351 115 (SEQ. ID NO.:513) VPLRPMTYK HIV-1 NEF 74-82 115 (SEQ. IDNO.:514) AIFQSSMTK HIV-1 gag p24 115 (SEQ. ID NO.:515) 325-333QVPLRPMTXTK HIV-1 nef 73-82 118 (SEQ. ID NO.:516) TTNYTIFKHCV NSI238-246 114 (SEQ. ID NO.:517) AAVDLSHFLKEK HIV-1 nef 83-94 120 (SEQ. IDNO.:518) ACQGVGGPGG HIV-1 II 1B p24 122 HK 349-359 (SEQ. ID NO, :519)HLA-A24 SYLDSGIHF* β-catenin, mutated 123 (proto-onocogen) (SEQ. TDNO.:520) 29-37 T cell epitopes RYLKDQQLLHIV GP 41 583-591 124 (SEQ. IDNO.:521) AYGLDFYIL P15 melanoma 125 (SEQ. ID NO.:522) Ag 10-18AFLPWHIRLFL Tyrosinase 206-215 126 (SEQ. ID NO.:523) AFLPWIHRLFTyrosinase 206-214 126 (SEQ. ID NO.:524) RYSIFFDY Ebna-3 246-253 101(SEQ. ID NO.:525) T cell epitope ETINEEAAEW HIV-1 gag p24 127 (SEQ. IDNO.:526) 203-212 T cell epitopes STLPETTVVRRHBV cAg 141-151 129 (SEQ. IDNO, :527) MSLQRQFLR ORF 3P-gp75 130 (SEQ. ID NO.:528) 294-321 (bp)LLPGGRPYR TRP (tyrosinase rel.) 131 (SEQ. ID NO.:528) 197-205 T cellepitope IVGLNKIVRHIV gag p24 267-267-275 132, (SEQ. ID NO.:530) 133AAGIGILTV Melan A/Mart- 127 35 100 (SEQ. ID NO.:531)

[0102] Table III sets forth additional antigens useful in the inventionthat are available from the Ludwig Cancer Institute. The Table refers topatents in which the identified antigens can be found and as such areincorporated herein by reference. TRA refers to the tumor-relatedantigen and the LUD No. refers to the Ludwig Institute number. TABLE IIILUD Patent Peptide TRA No. No. Date Patent Issued (Antigen) HLA MAGE-45293 5,405,940 11 April 1995 EVDPASNTY HLA-A1 (SEQ. ID NO.:532) MAGE-415293 5,405,940 11 April 1995 EVDPTSNTY HLA-A I (SEQ ID NO:533) MAGE-55293 5,405,940 11 April 1995 EADPTSNTY HLA-A I (SEQ ID NO:534) MAGE-515293 5,405,940 11 April 1995 EADPTSNTY HLA-A I (SEQ ID NO:534) MAGE-65294 5,405,940 11 April 1995 EVDPIGHVY HLA-A1 (SEQ ID NO:535) 5299.25,487,974 30 January 1996 MLLAVLYCLL HLA-A2 (SEQ ID NO:536) 53605,530,096 25 June 1996 MLLAVLYCL HLA-B44 (SEQ ID NO:537) Tyrosinase5360.1 5,519,117 21 May 1996 SEIWRDIDFA HLA-B44 (SEQ ID NO:538)SEIWRDIDF (SEQ ID NO:539) Tyrosinase 5431 5,774,316 28 April 1998XEIWRDIDF HLA-B44 (SEQ ID NO:540) MAGE-2 5340 5,554,724 10 September1996 STLVEVTLGEV HLA-A2 (SEQ ID NO:541) LVEVTLGEV (SEQ ID NO:542)VIFSKASEYL (SEQ ID NO:543) IIVLAIIA1 (SEQ ID NO:544) KIWEELSMLEV (SEQ IDNO:545) LIETSYVKV (SEQ ID NO:546) 5327 5,585,461 17 December 1996FLWGPRALV HLA-A2 (SEQ ID NO: 547) TLVEVTLGEV (SEQ ID NO:548) ALVETSYVKV(SEQ ID NO:549) MAGE-3 5344 5,554,506 10 September 1996 KIWEELSVL HLA-A2(SEQ ID NO:550) MAGE-3 5393 5,405,940 11 April 1995 EVDPIGHLY HLA-A1(SEQ ID NO:551) MAGE 5293 5,405,940 11 April 1995 EXDX5Y HLA-A1 (SEQ. IDNO.:552) (but not EADPTGHSY) (SEQ. ID NO.:553) E (A/V) D X5 Y (SEQ. IDNO.:554) E (A/V) D P X4 Y (SEQ. ID NO.:555) E (A/V) D P (I/A/T) X3 Y(SEQ. ID NO.:556) E (A/V) D P (I/A/T) (G/S) X2 Y (SEQ. ID NO.:557) E(A/V) D P (I/A/T) (G/S) (H/N) X Y E (A/V) DP (I/A/T) (G/S) (H/N) (L/T/V)Y (SEQ. 11) NO.:559) MAGE-1 5361 5,558.995 24 September 1996ELHSAYGEPRKLLTQD HLA-C (SEQ ID NO:560) Clone 10 EHSAYGEPRKLL (SEQ IDNO:561) SAYGEPRKL (SEQ ID NO:562) MAGE-1 5253.4 TBA TBA EADPTGHSY HLA-AI (SEQ ID NO:563) (SEQ ID NO:564) Clone 10 MAARAVFLALSAQLLQ HLA-C (SEQID NO:565) Clone 10 AARAVFLAL HLA-C (SEQ ID NO:566) Clone 10 GAGE 5323.25,648,226 15 July 1997 YRPRPRRY HLA- CW6 (SEQ. ID NO.:567) BAGE 5310.1TBA TBA MAARAVFLALSAQLLQARLMKE HLA-C

[0103] Preferred peptide antigens are those of tumor associated antigens(TAA) and chronic infections. Particularly preferred peptide antigensare derived from tyrosinose, gp100 or Melan A for the treatment ofmelanoma.

[0104] The peptide antigens of this invention are readily prepared usingstandard peptide synthesis means known in the art. Generally they can beprepared commercially by one of numerous companies that do chemicalsynthesis. An example is American Peptides, Inc., where the distributoris CLINALFA AG (Laufelfingen, Switzerland). The antigens are prepared inaccordance with GMP standards. Purity is assessed by analytical HPLC.The product is characterized by amino-acid analysis and tested forsterility and the absence of pyrogens.

[0105] In delivering an appropriate antigen, e.g., a polypeptide, to theanimal's system it may be delivered directly as the polypeptide, or itmay be delivered indirectly, e.g., using a DNA construct or vector, or arecombinant virus that codes for the desired antigen. Any vector drivingexpression in a professional antigen presenting cell is suitable forthis purpose. In the indirect delivery, the antigen is expressed in thecell, to be presented by the MHC Class I on the surface of the cell tostimulate the CTL response.

[0106] In a preferred embodiment of the invention an encoded antigen canbe delivered in the form of a naked plasmid expression vector.Particularly useful constructs are disclosed in U.S. patent applicationSer. No. 09/561,572 entitled EXPRESSION VECTORS ENCODING EPITOPES OFTARGET-ASSOCIATED ANTIGENS which is incorporated herein by reference inits entirety. The feasibility of and general procedures related to theuse of naked DNA for immunization are described in U.S. Pat. No.5,589,466, entitled “INDUCTION OF A PROTECTIVE IMMUNE RESPONSE IN AMAMMAL BY INJECTING A DNA SEQUENCE” and U.S. Pat. No. 5679647, entitled“METHODS AND DEVICES FOR IMMUNIZING A HOST AGAINST TUMOR-ASSOCIATEDANTIGENS THROUGH ADMINISTRATIONS OF NAKED POLYNUCLEOTIDES WHICH ENCODETUMOR-ASSOCIATED ANTIGENIC PEPTIDES” which are herein incorporated byreference in their entirety. However the former teaches onlyintramuscular or intradermal injection while the latter teaches onlyadministration to skin or mucosa. Administration directly to thelymphatic system is greatly more efficient (see examples 6-9, below).Single bolus injection into lymph node required only 0.1% of the doserequired in order to obtain a similar level of CTL response byintramuscular (i.m.) injection. It is therefore feasible to establish aprotective response against systemic viral infection with a single bolusdelivered i.ln., but not with a dose nearing the practical limitdelivered i.m. Even repeated bolus injections i.m. failed to establish aprotective response against a peripheral virus infection or transplantedtumor whereas lower doses administered i.m. were completely effective.

[0107] In another embodiment of the invention an encoded antigen can bedelivered in the form of a viral vector. A wide array of viruses withmodified genomes adapted to express interposed reading frames but oftenno, or at least a reduced number of, viral proteins are known in theart, including without limitation, retroviruses including lentiviruses,adenoviruses, parvoviruses including adeno-associated virus,herpesviruses, and poxviruses including vaccinia virus. Such viralvectors facilitate delivery of the nucleic acid component into the cellallowing for expression. A subset of these vectors, such as retrovirusesand parvoviruses, also promotes integration of their nucleic acidcomponent into the host genome, whereas others do not.

[0108] Bacteria can also serve as vectors, analogously to viruses, i.e.they can be used to deliver a nucleic acid molecule capable of causingexpression of an antigen. For example, a strain of Listeriamonocytogenes has been devised that effects its own lysis upon enteringthe cytosol of macrophages (its normal target), thereby releasingplasmid from which antigen was subsequently expressed (Dietrich, G. etal. Biotechnology 16:181-185, 1998 which is herein incorporated byreference in their entirety). Shigela flexneri and Escherichia coli havebeen similarly used (Sizemore, D. R. et al. Science 270:299-302, 1995,and Courvalin, P. et al. Life Sci. 318:1207-1212, 1995, respectively,which are herein incorporated by reference in their entirety).

[0109] The use of microbial vectors for nucleic acid delivery can becomplicated by the immune reactions the vectors themselves provoke. Whenprolonged or repeated administration is required, antibody elicited bythe earlier treatment can prevent useful quantities of the vector fromever reaching its intended host. However, by direct administration into,for example, a lymph node, the combination of proximity to host cellsand the much reduced effective dose makes it possible to administer adose capable of evading or overwhelming an existing antibody titer.

[0110] The word vector has been used, here and elsewhere, in referenceto several modalities and variously modified (e.g. expression vector,viral vector, delivery vector, etc.). The underlying principle here isthat a nucleic acid capable of causing expression of an antigenultimately arrives in an APC, rather than the antigen itself. Unlessmodified, explicitly or by local context, wherever the term vector isused herein, it is intended to encompass all such possibilities.

[0111] These foregoing techniques are distinct from the approach ofmodifying the microbial genome (including extra-chromosomal DNA) so thatthe antigen is produced as a component of the microbe (virus, bacteria,fingi, protazoan, etc., etc.), which is then itself administered as theimmunogen. Obviously, the genomic modification would most likely involvethe use some reagent falling within the above definition of vector.Again, the distinction is whether the vaccine includes the alreadysynthesized antigen, or a nucleic acid capable of causing an APC toexpress the antigen in vivo. This strategy constitutes a furtherembodiment of the invention. For rhetorical clarity we have discussedthese approaches as if they were mutually exclusive, but in factcombinations are possible, e.g., a virus vector as above that alsoincorporates a target epitope into a capsid or envelope protein.

[0112] Similarly, antigen presenting cells can also be manipulated invitro and then themselves used as the active component of a vaccine.Antigen expression can be conferred by delivering nucleic acid encodedantigen using any of the transduction techniques known in the art,including without limitation electroporation, viral or bacterialtransduction, lipid-mediated transduction, and biolistic bombardment.Alternatively the APCs may simply be pulsed with antigen. As with any ofthe other embodiments of this invention an antigen can be anapproximately 8-10 amino acid peptide representing a single epitope, acomplete protein, a polypeptide encompassing one or more epitopes(including epitopes originally derived from multiple proteins) or otherforms of antigen described above.

[0113] Antigens may be used alone or may be delivered in combinationwith other antigens or with other compounds such as cytokines that areknown to enhance immune stimulation of CTL responses, such as, GM-CSF,IL-12, IL-2, TNF, IFN, IL-18, IL-3, IL-4, IL-8, IL-9, IL-13, IL-10,IL-14, IL-15, G-SCF, IFN alpha, IFN beta, IFN gamma, TGF alpha, TGFbeta, and the like. The cytokines are known in the art and are readilyavailable in the literature or commercially. Many animal and humantumors have been shown to produce cytokines such as IL-4, IL-10, TGF-Bthat are potent modulators of the immune response and that protecttumors from immune-mediated destruction. The production of IL-4, IL-10or TGF-B by the tumors may achieve this protective effect by suppressingthe induction of cellular immunity, including the elaboration of CTLresponses. Alternatively, cytokines that support CTL responses can beexogenously added to help in the balance between induction of anti-tumorcell mediated and non-tumor-destructive humoral responses. Several suchexogenous cytokines show utility in experimental mouse vaccinationmodels which are known to enhance CTL responses, including GM-CSF, IFNand IL-2. An effective exogenous cytokine that may be used is GM-CSF.GM-CSF is reported to enhance the expression of the so called“co-stimulatory” molecules, such as B7-1 or B7-2 on antigen presentingcells (APC), which are important players in the symphony of interactionsthat occur during stimulation of CTL by APC. Moreover, GM-CSF is knownto induce activation of APC and to facilitate growth and differentiationof APC, thereby making these important CTL stimulating cells availableboth in greater numbers and potency.

Delivery of the Antigen

[0114] This invention is based in part on the observation that a CTLresponse is not sustained using standard vaccine techniques. While notwanting to be bound by any particular theory, it is thought that T cellsdo not have a functional memory that is long-lived. Antibody-mediatedB-cell memory, on the other hand, appears to have a long-lived effectormemory. Thus, delivering an antigen that produces a CTL response must bedone over time to keep the patient's immune system appropriatelystimulated to attack the target cells. While it has been suggested thatantigens and adjuvants can be prepared as biodegradable microspheres orliposomes, none of these preparations have thus far provided a CTLresponse that is useful for attacking cancer cells or pathogens on along term basis. The delivery must be sustained over the desired periodof time at a level sufficient to maintain the antigen level to obtainthe desired response and that it must be delivered from a reservoirhaving fluid antigen composition that is introduced so that it reachesthe animal's lymphatic system.

[0115] Ultimately antigen must find its way into the lymphatic system inorder to efficiently stimulate CTL. However, delivery of antigenaccording to the invention can involve infusion into variouscompartments of the body, including but not limited to subcutaneous,intravenous, intraperitoneal and intralymphatic, the latter beingpreferred. Each of these various points of infusion results in antigenuptake into the lymphatic system. The relative amounts of antigen neededto induce a beneficial CTL response varies according to the differentsites of infusion. In general, direct infusion of antigen into the lymphsystem is deemed to be the most efficient means of inducing a CTLresponse, but that the material difference between the various routes isnot necessarily relevant in terms of the quantity of antigen needed, or,in terms of the operating parameters of the invention. The pump systemsof the invention are capable of delivering material quantities ofantigen in a range that makes the invention suitable for inducing CTLresponse through delivery to all compartments of the body. CTLstimulation based on delivery of antigen via various routes will bevariable, based on the properties of different antigens, which willreflect factors that influence antigen behavior in the body and its rateof equilibration to (or longevity in) the lymph, such an antigenstability in the body fluid, solubility of antigen in body fluid,binding affinity for HLA and potency as a stimulator of CTL.

[0116] It is most efficient, and therefore, preferred, that theintroduction is done as directly as possible to the lymphatic system toavoid the destruction of the antigen by metabolism in the body. Whenintroduction of a fluid antigen composition occurs subcutaneously,larger quantities of antigen are needed to assure enough antigen reachesthe lymphatic system. Such subcutaneous injection is contemplated bythis invention if it can be justified by factors such as cost, stabilityof the antigen, how quickly the antigen gets to the lymph system, howwell it equilibrates with the lymph, and other factors that theattending doctor or specialist will recognize. Subcutaneous deliverywill generally require 100 to 1000 times more antigen than directdelivery to the lymph system. It is preferable, therefore, that theantigen composition is introduced through a device for localadministration to the lymphatic system, e.g. the spleen, a lymph node,or a lymph vessel. The device for local administration may be positionedoutside the patient or implanted into the patient. In either case, thedevice will have a reservoir to hold the fluid antigen-containingcomposition, a pump to transfer the composition, and a transmissionchannel leading from the reservoir to be directed to the preferredregion of administration in the patient's body. In either case it ispreferably portable.

[0117] For the device positioned outside the patient's body (theexternal device), there are numerous devices used for delivering insulinto diabetic patients that are useful in this invention. Generally theseare comprised of a reservoir for holding the antigen composition(instead of insulin), a programmable pump to pump the composition out ofthe reservoir, a transmission channel or line for transmitting thecomposition, and a means to introduce the composition into the animal'sbody to ultimately reach the lymphatic system.

[0118] The pump employed may be a roller/peristaltic pump, a syringepump, a piston/valve pump, a gas pressure pump, or the like that has apower source (generally a battery for portability) that is programmableto deliver the desired level of antigen composition to the patient'sbody and the lymphatic system. A further discussion of the operation ofthese pumps may be found “Insulin Pump Therapy” by E. Austenst and T.Stahl, Walter de Gruyter, Berlin, N.Y. (1990), at Chapter 3. A list ofpumps available at that time that are useful for this invention aregiven in Table IV.

[0119] More recent versions of these pumps are available from themanufacturers shown. TABLE IV Name Manufacturer/distributor Weight (g)Size (mm) Nordisk Infusor Nordisk 180 100 × 60 × 20 Betatron I CPI/Lilly197  99 × 66 × 20 RW 90 P/RW 91 Dahedi/EA Satorius 110 109 × 42 × 22P/RW 92 Instruments MRS 4-Infuser Disetronic 100  75 × 53 × 19 B-D 1000Becton-Dickinson 131 7  8 × 57 × 20 Nordisk Infusor Nordisk 180 113 × 65× 22 MK 11 MRS 3-Infuser Disetronic 100  75 × 53 × 18 A S8 MPAutosyringe/Travenol 161 102 × 64 × 19 Betatron 11 CPULilly 197  99 × 66× 20 Minimed 504 Pacesetter/Haselmeyer 106  86 × 21 × 51 Minimed 404 S*Pacesetter 106  86 × 21 × 51 MRS I/H-Tron Disetronic/Hoechst 100  75 ×53 × 18

[0120] Particularly useful pumps are the Disetronic H-Tron V 100 InsulinPump from Disetronic Medical Systems, Burgdorf, Switzerland and theMinimed 507 Insulin Pump from MiniMed Inc., 12744 San Fernando Road,Sylmar, Calif. 91342. The MiniMed is particularly useful in that itallows programming a bolus without looking at the pump through a seriesof audio tones (settable in either 0.5 or 1.0 unit increments) andallows programming a bolus for delivery over an extended period oftime—from 30 minutes to 4 hours. It provides up to 12 basal rates (orprofiles) that can be programmed per 24 hours from 0.0-25 units/hour in0. 1 unit increments. The device allows for the temporary increase ordecrease of a set basal rate from 30 minutes to 24 hours in 30 minuteincrements, Other features relating to safety, time display, memory,etc. are available from the manufacturer.

[0121] The reservoir for the antigen composition should be large enoughfor delivery of the desired amount of antigen over time and is easilyrefillable or replaceable without requiring the user to reinsert themeans for introducing the antigen composition to the lymph system.

[0122] In preparing the antigen compositions of this invention, acomposition (preferably aqueous) is prepared to be compatible with thelymph system and is physiologically acceptable to the animal beingtreated. In preparing the antigen compositions useful in this inventionone considers the physicochemical properties of the antigen such as theisoelectric point, molecular weight, glycosylation or otherpost-translational modification, and overall amino acid composition.These properties along with any known behavior of the drug in differentsolutions (e.g. different buffers, cofactors, etc.) as well as its invivo behavior will help guide the choice of formulation components. Oneparameter that impacts all the major degradation pathways is thesolution pH. Thus, the initial formulations also assess the pHdependence of the degradation reactions and the mechanism fordegradation can often be determined from the pH dependence to determinethe stability of the protein in each solution. Rapid screening methodsusually involve the use of accelerated stability at elevatedtemperatures (e.g. 40° C.) using techniques known in the art.

[0123] In general the antigen compositions useful in this invention willbe prepared suitable for parenteral injection, in very small quantities.As such a composition must be free of contamination and have a pHcompatible with the lymph system. However, because very small quantitiesof the antigenic composition will be delivered it need not be the samepH as blood or lymph, and it need not be aqueous-based. For antigensthat are less soluble a suitable cosolvent or surfactant may be used,such as dimethyl sulfoxide (DMSO) or PLURONIC brand surfactants. The pHrange that is compatible is from about 6.7-7.3 and can be prepared usingwater for injection to meet USP specifications (see Remington: TheScience and Practice of Pharmacy, Nineteenth Edition; Chapters 86-88).Generally, a standard saline solution that is buffered with aphysiologically acceptable weak acid and its base conjugate, e.g., aphosphate or citrate buffering system, will be the basis of the antigencomposition. In some cases, a small amount of an antioxidant may beuseful to stabilize the composition and prevent oxidation. Factors toconsider in preparing the antigen compositions may be found in the 1994American Chemical Society book entitled “Formulation and Delivery ofProteins and Peptides” (Acs Symposium Series, No. 567) by Jeffery L.Cleland and Robert Langer (Editor)).

[0124] For nucleic acid encoded antigens similar considerations apply,although the variety of physico-chemical properties encountered withpolypeptides is absent, so that acceptable formulations will have nearlyuniversal applicability. As seen in examples 6-10, plasmid DNA instandard phosphate buffered saline (PBS) is an acceptable and effectiveformulation. In some embodiments of the invention, DNA is administeredcontinuously or intermittently at short intervals, from a reservoir wornon, or implanted in, the patient's body. It is preferable that the DNAbe maintained in a soluble, stable form at or near body temperature overa period of time measured minimally in days. In such applications wherethe formulated nucleic acid will be delivered from a reservoir over aperiod several days or longer, the stability of the nucleic acid at roomor body temperature for that period of time, as well as its continuedsterility, take on increased importance. The addition of bacteriostaticagents (e.g. benzyl or ethyl alcohol) and chelating agents (e.g. EDTA)is useful toward these ends. Formulations containing about 1-10% ethylalcohol, 0-1% benzyl alcohol, 0.25-0.5 mM EDTA and a citrate-phosphatebuffer of pH 7.4-7.8 generally perform well. Such formulations are alsoappropriate for bolus injections.

[0125] Generally the amount of the antigen in the antigen compositionwill vary from patient to patient and from antigen to antigen, dependingon such factors as the activity of the antigen in inducing a responseand the flow rate of the lymph through the patient's system. In generalthe antigen composition may be delivered at a rate of from about 1 toabout 500 microliters/hour or about 24 to about 12000 microliters/day.The concentration of the antigen is such that about 0.1 micrograms toabout 10,000 micrograms of the antigen will be delivered during 24hours. The flow rate is based on the knowledge that each minuteapproximately about 100 to about 1000 microliters of lymph fluid flowsthrough an adult inguinal lymph node. The objective is to maximize localconcentration of vaccine formulation in the lymph system. A certainamount of empirical investigation on patients will be necessary todetermine the most efficacious level of infusion for a given vaccinepreparation in humans.

[0126] To introduce the antigen composition into the lymphatic system ofthe patient the composition is preferably directed to a lymph vessel,lymph node, the spleen, or other appropriate portion of the lymphsystem. Preferably, the composition is directed to a lymph node such asan inguinal or axillary node by inserting a catheter or needle to thenode and maintaining the catheter or needle throughout the delivery.Suitable needles or catheters are available made of metal or plastic(e.g. polyurethane, polyvinyl chloride [PVC], TEFLON, polyethylene, andthe like). In inserting the catheter or needle into the inguinal nodefor example, the inguinal node is punctured under ultrasonographiccontrol using a Vialon™ Insyte-W™ cannula and catheter of 24G3/4 (BectonDickinson, USA) which is fixed using Tegadenn transparent dressingTegaderm™ 1624, 3M, St. Paul, Minn. 55144, USA). This procedure isgenerally done by an experienced radiologist. The location of thecatheter tip inside the inguinal lymph node is confirmed by injection ofa minimal volume of saline, which immediately and visibly increases thesize of the lymph node. The latter procedure allows confirmation thatthe tip is inside the node and can be performed to ensure that the tipdoes not slip out of the lymph node can be repeated on various daysafter implantation of the catheter. In case the tip did in fact slip outof location inside the lymph node, a new catheter can be implanted.

[0127] In another embodiment, the antigen is delivered to the lymphaticsystem through an article of manufacture that is implanted in theanimal, preferably at or near a site of a lymphatic organ. The articlewill include a pump that can deliver the antigen at a controlled rateover a pre-determined period of time and is suitable for use in thehost. Several devices are known in the art for the delivery of agents(such as drugs) in humans or animals and these can be used or adaptedfor use in the present invention.

[0128] The implantable device will be similar to the external devicediscussed above in that it comprises a reservoir of aphysiologically-acceptable, aqueous, antigen-containing composition thatis capable of inducing a CTL response in an animal, a pump positioned inassociation with the reservoir to deliver the composition at a definedrate, a transmission channel to discharge the composition from thereservoir, and optionally a delivery line connected to the transmissionchannel, which delivery line is of a size suitable for positioning inthe animal and for delivery of the composition in a manner that reachesthe lymphatic system of the animal.

[0129] Preferably the pump in the implantable device is an osmotic pumpof the type used in the ALZET® model device or the DUROS™ model devicepioneered by Alza Corporation, Palo Alto, Calif. or in a device made byPharmetrix and exemplified in U.S. Pat. No. 4,838,862. The osmotic pumputilizes the osmotic effect using a membrane permeable to water butimpermeable to a solute. Osmotic pressure built up in a device is usedto deliver a composition at a controlled rate over time. A review byGiancarlo Santus and Richard Baker of “Osmotic Drug Delivery: A Reviewof the Patent Literature” in the Journal of Controlled Release 35 (1995)1-21, provides useful guidelines for the type of osmotic pumps that areuseful in this invention. The osmotic pump forces the compositionthrough a discharge orifice to discharge the composition, Optionally adelivery line connects to the discharge orifice to position the linesuitably for delivery to the lymphatic system of the animal. Patentsthat describe devices useful in this invention include the followingU.S. patents: (A) U.S. Pat. No. 3,604,417 assigned to American Cyanamid;(B) U.S. Pat. Nos. 4,838,862; 4,898,582; 5,135,498; 5,169,390; and5,257,987 all assigned to Pharmetrix, (C) U.S. Pat. Nos. 4,340,048;4,474,575; 4,552,651; 4,619,652; 4,753,651; 3,732,865; 3,760,804;3,760,805; 3,929,132; 3,995,632; 4,034,756; 4,350,271; 4,455,145;5,017,381; 5,023,088; 5,030,216; 5,034,229; 5,037,420; 5,057,318;5,059,423; 5,110,596; 5,110,597; 5,135,523; 5,137,727; 5,174,999;5,209,746; 5,221,278; 5,223,265; 3,760,984; 3,987,790; 3,995,631;4,203,440; 4,286,067; 4,300,558; 4,304,232; 4,340,054; 4,367,741;4,450,198; 4,855,141; 4,865,598; 4,865,845; 4,872,873; 4,929,233;4,963,141; 4,976,966, all assigned to Alza Corp. Each of the foregoingpatents are incorporated herein by reference.

[0130] A basic osmotic pump device incorporates a housing containing achamber for storing the antigen containing composition to be delivered,separated from a compartment containing an osmotic salt material by abarrier that is moveable under pressure such as a piston or a flexibleimpermeable membrane. The compartment containing the osmotic salt isseparated from osmotic fluid by a semipermeable membrane. In someembodiments, a fluid barrier, such as a foil sheet, isolates the osmoticsalt chamber from the osmotic fluid, keeping the pump inactivated untilremoval of the barrier immediately prior to use. Other osmotic pumpdevices use body fluid as the osmotic fluid. In these devices asemipermeable membrane separates the osmotic salt compartment from bodyfluids, and the pump is activated once inserted into the body underexposure to body fluids. In either case, volumetric expansion of theosmotic salt compartment drives the expulsion of the stored antigen fromthe compartment and into the surrounding environment of the body. Thesepumps have been highly successful at achieving steady-state pumping anddelivery of agents. The pumps are of a small size that can be insertedinto a patient, with flexible consideration as to location. This isimportant in the case of CTL vaccines, since the inventor has determinedthat efficient induction of CTL responses is contingent on the antigenor antigen expression system being delivered into the lymphatic system,in order to ultimately achieve antigen delivery into a lymphatic organsuch as the spleen. Antigen delivered into a lymph node is 100-1000times more efficient at inducing CTL responses compared withconventional subcutaneous delivery. A modification to the osmotic pumpincorporates a microcatheter attachment (i.e., the optional deliveryline) at its discharge end, such that when the pump is implantedproximal to a lymphatic organ, such as a lymph node, the catheter can beinserted into the organ to facilitate delivery of the vaccine directlyinto the lymphatic system.

[0131] Prior to the administration of the antigen using any of the abovevehicles, methods may be used to assist in the determination of theoptimum location for the antigen delivery. For example, when using theosmotic pump, radiography may be used to image a patient's lymphaticflow, to determine where relatively high lymphatic drainage occurs, inorder to decide upon an insertion position for the osmotic pump thatmaximizes delivery into the lymphatic system. Since each patient hasunique lymphatic drainage profiles, imaging would be conducted for eachindividual prior to insertion of osmotic pump for delivery of antigen.When using direct cannulation of the lymphatic vessel, such as in theuse of osmotic or insulin pumps to deliver antigen, ultrasound can beused to position the needle directly into the lymphatic vessel and tomonitor its positioning over the period of treatment.

[0132] The following non-limiting examples are illustrative of thepresent invention.

EXAMPLES Materials and Methods For Examples 1-5

[0133] Mice: The generation of T cell receptor transgenic mice (TCR+mice) in which approx. 90% of the CD8+ T cells express a TCR recognizingthe immunodominant LCMV-glycoprotein epitope (gp-peptide aa33-41,p33:K.AVYNFATC-SEQ ID NO:569) presented on H-2D^(b), has been describedin detail. All experimental mice were between 8 and 12 weeks of age andbred and held under strict pathogen free conditions at the Institut FürLabortierkunde at the University of Zurich.

[0134] Viruses: LCMV (Armstrong strain) was originally obtained from Dr.M. B. A. Oldstone, Scripps Clinics and Research Foundation, LaJolla, SanDiego, Calif. Seed virus was grown on BHK cells and plaqued on MC57cells using an immunological focus assay, as described previously.

[0135] Osmotic pump: ALZA model #1007b.

[0136] In vivo protection assays for specific CTL activity: The in vivoassay for the detection of CTL activity by challenge infections withLCMV has been described in detail previously (Oehen et al. 1991).Briefly, mice are intravenously challenged with 2×10³ pfu of LCMV(Armstrong), After 4 days the titer of LCMV is determined using theabove mentioned immunological focus assay.

[0137] Primary ex vivo cytotoxicity against LCMV-gp: Mice were injectedintravenously with 10 μg of p33. After 36 hours spleen single cellsuspensions were coincubated for 5 h with ⁵¹Cr-labeled syngeneic EL-4(H-2^(b)) target cells, that were either pulsed with p33 or leftunpulsed. Specific lysis was calculated as [(experimental ⁵¹Crrelease−spontaneous ⁵¹Cr release)/(total ⁵¹Cr release−spontaneous ⁵¹Crrelease)×100%].

[0138] LCMV induced foot pad swelling reaction: Mice were infected withLCMV (Armstrong) by intradermal injection into the hind footpad (5000pfu in 30:1), Footpad thickness was measured daily with a spring loadedcaliper. Footpad swelling is calculated as (measured thickness-thicknessbefore injection)/(thickness before injection).

Example 1 Continuous Release of Peptide Antigen Using Osmotic PumpInduces Potent CTL Response in C5BL/6 Mice

[0139] C57BL/6 mice were either intravenously injected with a singledose of 50 μg p33 (including 500 ng GM-CSF) (circles) or were implantedwith a micro-osmotic pump releasing a mixture of 50 μg of p33 and 500 ngGM-CSF over a time period of 7 days (triangles), or were left naive(data not shown). After 7 days mice were sacrificed to prepare singlecell suspensions from the spleen. Spleen cells were restimulated invitro for 5 days by p33 pulsed in the presence of low amounts of IL-2.Specific cytotoxicity was measured using ⁵Cr-labeled EL-4 target cellspulsed with p33. Specific lysis of EL-4 target cells without p33 wasless than 16% for all effectors. The results are shown in FIG. 1.

Example 2 Continuous Release of Antigen Induces CTL Immunity AgainstVirus in C57BL/6 Mice

[0140] C57BL/6 mice were either intravenously injected with a singledose of 50 μg p33 (including 500 ng GM-CSF. Pharmingen) or wereimplanted with a microsomotic pump releasing a mixture of 50 μg of p33and 500 ng GM-CSF over a time period of 7 days, or were left naive.After 7 days specific CTL activity was assessed in vivo using anti-viralprotection assays. C57BL/6 mice were intravenously challenged with LCMVArmstrong strain (2×10³ p.f.u.). After 4 days mice were sacrificed andLCMV titers were determined in spleens using an immunological focusassay. Mice implanted with osmotic pump showed significantly lower virustiters indicating active CTL immunity against the virus (Table V). TABLEV C57BL/6 Mice Virus Titer (loglo) Single injection 4.2 Single injection4.6 Single injection 4.0 Pump delivered 2.2 Pump delivered 1.8 Pumpdelivered 2.0 Unprimed 4.8 Unprimed 3.8 Unprimed 4.4

Example 3 Continuous Release of Antigen Maintains Potent CTL Effectorsin TCR Transgenic Mice

[0141] TCR transgenic mice were either intravenously injected with asingle dose of 50 μg p33 (circles) or were implanted with a microsomoticpump releasing a mixture of 50 μg of p33 (triangles), or left naïve(squares). After 36 hours mice were sacrificed to prepare single cellsuspensions from the spleen which were assayed ex vivo for p33-specificcytotoxicity using ⁵¹Cr-labeled EL-4 target cells pulsed with p33.Similarly mice were either intravenously injected with a single dose of50 μg p33 (circles) or were implanted with a micro-osmotic pumpreleasing a mixture of 50 μg of p33 over a time period of 7 days(triangles), or were left naïve (squares). After 7 days mice weresacrificed to prepare single cell suspensions from the spleen to assayex vivo p33-specific cytotoxicity using ⁵¹Cr-labeled EL-4 target cellspulsed with p33. Specific lysis of EL-4 target cells without p33 wasless than 18% for all effectors. The results are shown in FIGS. 2A and2B.

Continuous Release of Antigen Maintains Protective CTL Response AgainstVirus Infection

[0142] After 7 days TCR transgenic mice were challenged by intradermalLCMV injection into their hind foot pads (2×10³ pfu in 30 μ). Theabsence of a foot pad swelling reaction, as observed in mice with animplanted pump (triangles), indicates that at the time point ofinjection there was active CTL immunity inhibiting local replication ofthe virus in the foot pad. In contrast, foot pad swelling, as observedin mice injected with the peptide as a single bolus (circles) and naivecontrol mice (data not shown), indicated that LCMV successfullyreplicated in the foot pad in the absence of protective CTL. The resultsare shown in FIG. 2C.

Example 4 Direct Delivery of Antigen into Lymphatic Organ DramaticallyIncreases Efficiency of CTL Induction

[0143] TCR transgenic mice were injected with graded doses of gp-peptidep33 either subcutaneously (S.C.), intravenously (I.V.) or directly intothe spleen (I.S.) via a small abdominal incision. The efficiency of CTLinduction was assessed by measuring gp-specific CTL activity 24 hoursafter injection. CTL activity is known to peak one day after injectionof peptide. Mice were sacrificed to prepare single cell suspensions fromdraining lymph nodes or from spleen to assay ex vivo p33-specificcytotoxicity using ⁵¹Cr-labeled EL-4 target cells pulsed with p33.Specific lysis of EL-4 target cells without p33 was less than 12% forall effectors. The results are shown in FIG. 3.

Example 5 Dendritic Cells Purified from Mice Receiving IntrasplenicInjection of Peptide Potently Stimulate CTL

[0144] The effect of directing peptide delivery into lymphatic systemwas assessed. Peptide p33 was injected either i.v., s.c. or directlyinto the spleen of wild-type C57BL/6 mice. After 2 hours, DCs wereisolated from the spleen of animals injected either i.s. or i.v., andadditionally from the regional draining lymph nodes of animals injecteds.c. Cells isolated from these tissues were sorted for DCs usingmagnetic beads coupled with a monoclonal antibody recognizing theintegrin-alpha chain, a marker specific for DCs in spleen and lymphnodes. The positively and the negatively sorted cell fractions werecompared regarding their capacity to in vitro stimulate naive CD8+ Tcells from TCR transgenic mice specific for LCMV-gp. Only when peptidehad been directly injected into the spleen, the DC containing cellfraction stimulated CTL to proliferate, as measured by ³H-thymidineuptake. This indicated that CTL induction after direct injection ofpeptide into lymphatic organs reflected efficient loading of DCs withpeptide. In contrast, the fraction depleted for DC did not induceproliferation and DCs isolated from lymphoid organs of i.v. and s.cinjected mice were not effective stimulators. The results are shown inFIG. 4.

[0145] While the present invention has been described with reference towhat are presently considered to be the preferred examples, it is to beunderstood that the invention is not limited to the disclosed examples.To the contrary, the invention is intended to cover variousmodifications and equivalent arrangements included within the spirit andscope of the appended claims.

[0146] All publications, patents and patent applications are hereinincorporated by reference in their entirety to the same extent as ifeach individual publication, patent or patent application wasspecifically and individually indicated to be incorporated by referencein its entirety.

Example 6 Induction of CTL Response with Naked DNA is Most Efficient byIntra-lymph Node Immunization

[0147] In order to quantitatively compare the CD8+ CTL responses inducedby different routes of immunization we used a plasmid DNA vaccine(pEGFPL33A) containing a well-characterized immunodominant CTL epitopefrom the LCMV-glycoprotein (G) (gp33; amino acids 33-41) (Oehen, S., etal. Immunology 99, 163-169 2000), as this system allows a comprehensiveassessment of antiviral CTL responses. Groups of 2 C57BL/6 mice wereimmunized once with titrated doses (200-0.02 μg) of pEGFPL33A DNA or ofcontrol plasmid pEGFP-N3, administered i.m. (intramuscular), i.d.(intradermal), i.spl. (intrasplenic), or i.ln. (intra-lymph node).Positive control mice received 500 pfu LCMV i.v. (intravenous). Ten daysafter immunization spleen cells were isolated and gp33-specific CTLactivity was determined after secondary in vitro restimulation. As shownin FIG. 6, i.m. or i.d. immunization induced weakly detectable CTLresponses only when high doses of pEFGPL33A DNA (200 μg) wereadministered. In contrast, potent gp33-specific CTL responses wereelicited by immunization with only 2 μg pEFGPL33A DNA i.spl. and with aslittle as 0.2 μg pEFGPL33A DNA given i.ln. (FIG. 6; symbols representindividual mice and one of three similar experiments is shown).Immunization with the control pEGFP-N3 DNA did not elicit any detectablegp33-specific CTL responses (data not shown).

Example 7 Intra-lymph Node Immunization is the Most Efficient way toInduce Antiviral Anamnestic CTL Responses

[0148] Similar thresholds for CTL detection were observed when adifferent readout system was used. Groups of 2 C57BL/6 mice wereimmunized once with titrated doses of pEFGPL33A DNA (0.2-200 μg) andpositive control mice received 500 pfu LCMV i.v., as above. Ten dayslater they were challenged with 5×10⁴ pfu LCMV i.v. Four days afterchallenge spleen cells were isolated and ex vivo CTL activity wasassayed. This time point is too early to detect any primary CTL responseto LCMV infection in naïve mice (FIG. 7, Controls), but it allows thedetection of anamnestic CTL responses in mice which have been previouslyimmunized (FIG. 7, LCMV). As before, mice immunized with 200 μgintramuscularly showed only weak anamnestic CTL responses following LCMVchallenge, which were not detectable when lower immunizing doses of DNAwere used (FIG. 7). Those immunized by the i.spl. route showed strongananmestic CTL responses which titered out at an immunizing dose of 2 μgpEFGPL33A DNA, while the i.ln. route of immunization was again moreefficient with anamnestic CTL responses detectable when only 0.2 μgpEFGPL33A DNA was administered (FIG. 7).

[0149] These results from examples 6 and 7 clearly demonstrate thatadministration of plasmid DNA directly into lymphoid tissues is 100-to1000-fold more efficient than intradermal or intramuscular routes forthe induction of CTL responses. In addition, they show that theintra-lymph node route is around 10-fold more efficient than theintrasplenic route.

Example 8 Naked DNA Elicits Superior Protection Against Systemic andPeripheral Virus Infection by Intra-lymph Node Compared to IntramuscularImmunization

[0150] To examine whether the enhanced CTL responses elicited followingi.ln. immunization with plasmid DNA were able to qualitatively influenceantiviral immunity, we used challenge infections with LCMV or withrecombinant vaccinia virus expressing the LCMV-G (Vacc-G2) as models ofsystemic and peripheral virus infection, respectively. When systemicantiviral immunity was assessed by challenging the immunized mice(groups of 3 C57BL/6 mice) with a high dose of LCMV i.v. (500 pfu), micewhich had been immunized once with 200 μg pEGFPL33A DNA i.m. showed onlypartial and incomplete protection against systemic LCMV challenge, whilethose which had received 20 μg of pEFGPL33A DNA by the i.spl. or i.ln.routes were completely protected (FIG. 8A).

[0151] Eradication of Vacc-G2 infection from peripheral organs such asovaries, is dependent upon the presence of high levels of recentlyactivated effector CD8+ T cells (Kündig, T. M. et al. Proc. Natl. Acad.Sci. USA 93, 9716-9723, 1996; Bachmann, M. F., et al. Proc. Natl. Acad.Sci. USA 94, 640-645, 1997). Groups of 3 C57BL/6 mice were immunizedfour times at 6 day intervals with pEFGPL33A DNA administered eitheri.m. (100 μg per immunization) or i.ln. (10 μg per immunization). Fivedays after the last immunization they were challenged with 5×10⁶ pfuVacc-G2 i.p. and vaccinia titers in ovaries were assessed after afurther 5 days. Repeated i.m. immunization with pEFGPL33A DNA had noinfluence on the growth of Vacc-G2 in peripheral tissues (FIG. 8B). Incontrast, mice which were repetitively immunized with pEFGPL33A DNA bythe i.ln. route were completely protected against peripheral infectionwith Vacc-G2 (FIG. 8B).

[0152] These results illustrate that although repeated i.m. immunizationwith naked DNA induced detectable CTL responses, these were never ofsufficient magnitude to offer protection against virus infection. Incontrast, immunization with 10-fold lower amounts of DNA directly intolymphoid organs elicited quantitatively and qualitatively stronger CTLresponses, which gave complete protection against systemic or peripheralvirus challenge.

Example 9 Intra-lymph Node DNA Immunization Elicits Anti-tumor Immunity

[0153] To examine whether the potent CTL responses elicited followingi.ln. immunization were able to confer protection against peripheraltumors, groups of 6 C57BL/6mice were immunized three times at 6-dayintervals with 10 μg of pEFGPL33A DNA or control pEGFP-N3 DNA. Five daysafter the last immunization small pieces of solid tumors expressing thegp33 epitope (EL4-33) were transplanted s.c. into both flanks and tumorgrowth was measured every 3-4d. Although the EL4-33 tumors grew well inmice that had been repetitively immunized with control pEGFP-N3 DNA(FIG. 9), mice which were immunized with pEFGPL33A DNA i.ln. rapidlyeradicated the peripheral EL4-33 tumors (FIG. 9).

Example 10 Differences in Lymph Node DNA Content Mirrors Differences inCTL Response Following Intra-lymph Node and Intramuscular Injection

[0154] pEFGPL33A DNA was injected i.ln. or i.m. and plasmid content ofthe injected or draining lymph node was assessed by real time PCR after6, 12, 24, 48 hours, and 4 and 30 days. At 6, 12, and 24 hours theplasmid DNA content of the injected lymph nodes was approximately threeorders of magnitude greater than that of the draining lymph nodesfollowing i.m. injection. No plasmid DNA was detectable in the draininglymph node at subsequent time points (FIG. 10). This is consonant withthe three orders of magnitude greater dose needed using i.m. as comparedto i.ln. injections to achieve a similar levels of CTL activity.CD8^(−/−) knockout mice, which do not develop a CTL response to thisepitope, were also injected i.ln. showing clearance of DNA from thelymph node is not due to CD8+ CTL killing of cells in the lymph node.This observation also supports the conclusion that i.ln. administrationwill not provoke immunopathological damage to the lymph node.

Example 11 Stability of Plasmid in Different Formulations

[0155] DNA is a relatively stable molecule in the kind of formulationsof interest to test and thus little loss of material would be noted ifthe total amount of DNA were to be measured. Instead, the ratio ofsupercoiled to open-circle DNA was measured. Since a single nickanywhere in either strand of the DNA molecule will allow a supercoiledplasmid to relax to an open circle conformation this is an exquisitelysensitive indication of damage to the DNA backbone. Plasmid wasformulated, placed in vials in triplicate and incubated at 37° C. After1, 3 and 7 days aliquots were removed, subjected to anion exchange HPLC,and the peak areas corresponding to supercoiled and open-circle DNAcompared (see FIG. 1). Nine formulations were tested:

[0156] 1. 10% Ethanol, 0.25 mM EDTA, Citrate Phosphate pH 7.6

[0157] 2. 10% Ethanol, 0.25 mM EDTA,CitratePhosphate pH 7.4

[0158] 3. 1% Ethanol, 0. 5 mM EDTA, Citrate Phosphate pH 7.4

[0159] 4. 1% Ethanol, 0.5 mM EDTA, 1X PBS pH 7.4

[0160] 5. 0.5% Benzyl Alcohol, 0.25 mM EDTA, Citrate Phosphate pH 7.6

[0161] 6. 1% Benzyl Alcohol, 1% Ethanol, 0.5 mM EDTA, Citrate PhosphatepH 7.6

[0162] 7. 1% Benzyl Alcohol, 1% Ethanol, 0.5 mM EDTA, 0.1M TRIS pH 7.4

[0163] 8. 1% Benzyl Alcohol, 1% Ethanol, 0.5mM EDTA, 0.1MTRIS pH 8.2

[0164] 9. 1% Benzyl Alcohol, 1X PBS pH 8.2

[0165] Citrate Phosphate Buffer pH. 7.4 was made by mixing 9.15 parts(by volume) of 0.1M citric acid with 90.85 parts of 0.2M SodiumPhosphate Dibasic. Citrate Phosphate Buffer pH. 7.6 was made by mixing6.35 parts (by volume) of 0.1M citric acid with 93.65 parts of 0.2MSodium Phosphate Dibasic. These solutions were then added to the othercomponents to create a 2×buffer which was mixed with a equal volume ofDNA in water. Thus the final concentrations of citrate and phosphate inthe above buffers was on the order of 3 mM and 90 mM, respectively.

[0166] Formulations 1-3 and 6 gave superior results (see FIG. 11).

Example 12 Stability of Formulated Plasmid in Operating MINIMED 407CInfusion Pumps

[0167] Using a modification (final concentrations of 0.1 M sodiumphosphate dibasic and 0.05 M citric acid; pH 7.6±0.2) of formulation 6above, aliquots of 80, 160, and 320 μg DNA/ml were prepared and loadedin triplicate into MINIMED 3.0 reservoir syringes. A 200 μl sample wasdispensed and the reservoir syringes were inserted into MINIMED 407Cinfusion pumps and assembled with SILHOUETTE infusion sets fitted with3.1 mm catheters. The pump assemblies, set to dispense 10 μl/hour andwith the catheters inserted into collection vials, were placed in 37° C.incubators. At 4 and 8 days the catheters were briefly detached and 200μl bolus samples dispensed directly from the reservoir. Theconcentration of supercoiled DNA was determined for each sample by anionexchange HPLC and the use of a standard curve constructed with knownconcentrations of DNA. Plotting the resultant concentrations versus timeallows one to derive a slope indicating the rate of loss of supercoiledDNA. The average (of the triplicate samples) rates of loss were−0.056±1.88, 0.24±1.01, and 0.048±0.49 μg DNA/day for the 80, 160, and320 μg DNA/ml samples, respectively. None of these differ significantlyfrom zero.

Example 13 Administration of a DNA Plasmid Formulation of a TherapeuticVaccine for Melanoma to Humans

[0168] SYNCHROTOPE TA2M, a melanoma vaccine encoding HLA-A2-restrictedtyrosinase epitopes was formulated in 1% Benzyl alcohol, 1% ethylalcohol, 0.5 mM EDTA, citrate-phosphate, pH 7.6. Aliquots of 80, 160,and 320 μg DNA/ml were prepared for loading into MINIMED 407C infusionpumps. The catheter of a SILHOUETTE infusion set is placed into aninguinal lymph node visualized by ultrasound imaging. The assembly ofpump and infusion set was originally designed for the delivery ofinsulin to diabetics and the usual 17 mm catheter has been substitutedwith a 31 mm catheter for this application. The infusion set is keptpatent for 4 days (approximately 96 hours) with an infusion rate ofabout 25 μl/hour resulting in a total infused volume of approximately2.4 ml. Thus the total administered dose per infuision will beapproximately 200, 400, and 800 μg, respectively, for the threeconcentrations described above. Following an infusion subjects will begiven a 10 day rest period before starting a subsequent infusion. Giventhe continued residency of plasmid DNA in the lymph node afteradministration (as in example 10) and the usual kinetics of CTL responsefollowing disappearance of antigen, this schedule will be sufficient tomaintain the immunologic CTL response.

What is claimed is:
 1. A method of inducing and/or sustaining animmunological CTL response in a mammal, which method comprises:delivering an antigen in the form of a polypeptide directly to thelymphatic system of the mammal at a level sufficient to induce animmunologic CTL response in the mammal; and maintaining the level of theantigen in the mammal's lymphatic system over time sufficient tomaintain the immunologic CTL response.
 2. The method of claim 1, whereinsaid antigen is provided as an 8-10 amino acid peptide.
 3. The method ofclaim 1, wherein the peptide sequence is derived from a tumor-associatedantigen.
 4. The method of claim 3, wherein said tumor-associated antigenis selected from the group consisting of MelanA (MART-I), gp100 (Pmel17), tyrosinase, TRP-1, TRP-2, MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2,p15(58), CEA, RAGE, NY-ESO (LAGE), SCP-1, Hom/Mel-40, PRAME, p53, H-Ras,HER-2/neu, BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, Epstein Barrvirus antigens, EBNA, human papillomavirus (HPV) antigens E6 and E7,TSP-180, MAGE-4, MAGE-5, MAGE-6, p185erbB2, p180erbB-3, c-met, nm-23H1,PSA, TAG-72-4, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, β-Catenin, CDK4,Mum-1, p16, TAGE, PSMA, PSCA, CT7, telomerase, 43-9F, 5T4, 791Tgp72,alpha-fetoprotein , β-HCG, BCA225, BTAA, CA 125, CA 15-3 (CA27.29\BCAA), CA 195, CA 242, CA-50, CAM43, CD68\KP1, CO-029, FGF-5,G250, Ga733 (EpCAM), HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB/70K,NY-CO-1, RCAS1, SDCCAG16, TA-90 (Mac-2 binding protein\cyclophilinC-associated protein), TAAL6, TAG72, TLP, and TPS.
 5. The method ofclaim 1, wherein the peptide sequence is derived from a microbialantigen.
 6. The method of claim 1, wherein said antigen is provided as acomponent of a microorganism or mammalian cell.
 7. The method of claim6, wherein said microorganism is a protozoan.
 8. The method of claim 6,wherein said microorganism is a bacterium.
 9. The method of claim 6,wherein said microorganism is a virus.
 10. The method of claim 6,wherein said mammalian cell is an antigen presenting cell.
 11. Themethod of claim 10, wherein said antigen presenting cell is a dendriticcell.
 12. The method of claim 6, wherein said antigen is a nativecomponent of said microorganism or mammalian cell.
 13. The method ofclaim 6, wherein said microorganism or mammalian cell comprises anexogenous antigen.
 14. The method of claim 6, wherein said microorganismor mammalian cell comprises a recombinant nucleic acid encoding orpromoting expression of said antigen.
 15. The method of claim 13,wherein said microorganism or mammalian cell expresses atumor-associated antigen.
 16. The method of claim 13, wherein saidmicroorganism or mammalian cell expresses a microbial antigen native toa second microbial species.
 17. The method of claim 13, wherein saidantigen is provided as an 8-10 amino acid peptide.
 18. A method ofinducing and/or sustaining an immunological CTL response in a mammal,which method comprises: delivering an antigen, in the form of a vectorcomprising a nucleic acid encoding the antigen, directly to thelymphatic system of the mammal at a level sufficient to induce animmunologic CTL response in the mammal; and maintaining the level of theantigen in the mammal's lymphatic system over time sufficient tomaintain the immunologic CTL response.
 19. The method of claim 18,wherein the vector comprises a plasmid.
 20. The method of claim 19,wherein the vector further comprises a bacterium.
 21. The method ofclaim 20, wherein the bacterium is selected from the group consisting ofListeria, Shigella, Salmonella, and Escherichia.
 22. The method of claim18, wherein the vector further comprises a virus.
 23. The method ofclaim 22, wherein the virus is selected from the group consisting of poxviruses, adenoviruses, adeno-associated viruses, retroviruses, andherpesviruses.
 24. The method of claim 18, wherein said nucleic acidencodes a tumor-associated antigen.
 25. The method of claim 24, whereinsaid tumor-associated antigen is selected from the group consisting ofMelanA (MART-I), gp100 (Pmel 17), tyrosinase, TRP-1, TRP-2, MAGE-1,MAGE-3, BAGE, GAGE-1, GAGE-2, p15(58), CEA, RAGE, NY-ESO (LAGE), SCP-1,Hom/Mel-40, PRAME, p53, H-Ras, HER-2/neu, BCR-ABL, E2A-PRL, H4-RET,IGH-IGK, MYL-RAR, Epstein Barr virus antigens, EBNA, humanpapillomavirus (HPV) antigens E6 and E7, TSP-180, MAGE-4, MAGE-5,MAGE-6, p185erbB2, p180erbB-3, c-met, nm-23H1, PSA, TAG-72-4, CA 19-9,CA 72-4, CAM 17.1, NuMa, K-ras, β-Catenin, CDK4, Mum-1, p16, TAGE, PSMA,PSCA, CT7, telomerase, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein , β-HCG,BCA225, BTAA, CA 125, CA 15-3 (CA 27.29\BCAA), CA 195, CA 242, CA-50,CAM43, CD68\KP1, CO-029, FGF-5, G250, Ga733 (EpCAM), HTgp-175, M344,MA-50, MG7-Ag, MOV18, NB/70K, NY-CO-1, RCAS1, SDCCAG16, TA-90 (Mac-2binding protein\cyclophilin C-associated protein), TAAL6, TAG72, TLP,and TPS.
 26. The method of claim 18, wherein said nucleic acid encodes amicrobial antigen.
 27. The method of claim 26, wherein said antigen is aviral antigen.
 28. The method of claim 26, wherein said antigen is abacterial antigen.
 29. The method of claim 26, wherein said antigen is aprotozoal antigen.
 30. The method of claim 18, wherein said nucleic acidencodes a protein or other polypeptide.
 31. The method of claim 30,wherein said nucleic acid encodes an 8-10 amino acid peptide.
 32. Themethod of claim 18, wherein said nucleic acid is plasmid DNA in aformulation comprising about 1-10% ethyl alcohol, 0-1% benzyl alcohol,0.25-0.5 mM EDTA and a citrate-phosphate buffer of pH 7.4-7.8,comprising about 3-50 mM citrate and about 90 -200 mM phosphate.
 33. Themethod of claim 32, wherein said formulation comprises 1% ethyl alcohol,1% benzyl alcohol, 0.5 mM EDTA and a citrate-phosphate buffer of pH 7.4to 7.8 comprising 50 mM citrate and 100 mM phosphate.
 34. A method ofinducing and/or sustaining an immunological CTL response in a mammal,which method comprises: delivering a microorganism or mammalian celldirectly to the lymphatic system of the mammal at a level sufficient toinduce an immunologic CTL response in the mammal; and maintaining thelevel of the microorganism or mammalian cell in the mammal's lymphaticsystem over time sufficient to maintain the immunologic CTL response.35. A method of inducing and/or sustaining an immunological CTL responsein a mammal, which method comprises: delivering a nucleic acid capableof conferring antigen expression, directly to the lymphatic system ofthe mammal at a level sufficient to induce an immunologic CTL responsein the mammal; and maintaining the level of the nucleic acid in themammal's lymphatic system over time sufficient to maintain theimmunologic CTL response.
 36. A method of inducing and/or sustaining animmunological CTL response in a mammal, which method comprises:delivering a non-peptide antigen directly to the lymphatic system of themammal at a level sufficient to induce an immunologic CTL response inthe mammal; and maintaining the level of the antigen in the mammal'slymphatic system over time sufficient to maintain the immunologic CTLresponse.
 37. An article of manufacture for delivering an antigen thatinduces a CTL response in an animal, wherein the article is an externaldevice, and which article comprises: a reservoir of aphysiologically-acceptable, antigen-containing composition that iscapable of inducing a CTL response in an animal; a pump connected to thereservoir to deliver the composition at a defined rate; a transmissionline to discharge the composition from the reservoir; and, a deliveryline connected to the transmission line, which delivery line comprises acatheter of at least 20 mm for positioning in the animal and fordelivery of the composition to the lymphatic system of the animal.